Methods for covalently binding a cell surface protein and a ligand

ABSTRACT

The present invention relates to a method for covalently binding a cell surface protein and a ligand, the ligand being capable of specifically binding to the cell surface protein, the method consisting essentially of contacting the living cells expressing the cell surface protein with the ligand comprising at least one furan moiety, thereby covalently binding the cell surface protein and the ligand.

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 filing of International PatentApplication No. PCT/EP2017/054708, filed Mar. 1, 2017, which claims thebenefit of priority to European Patent Application No. 16158059.2, filedMar. 1, 2016. The entire contents of these applications are incorporatedherein by reference for all purposes.

FIELD OF THE INVENTION

The present invention is broadly in the field of cell-based assays fordetecting and/or identifying cell surface proteins or ligandsspecifically binding to cell surface proteins. In particular, theinvention concerns a method for covalently binding a cell surfaceprotein and a ligand. Further, the invention provides for cell-basedassays.

BACKGROUND OF THE INVENTION

Interactions between ligands, such as peptides or small molecules, andcell surface proteins are crucial for numerous key processes in livingorganisms. Approximately one third of the mammalian genome encodes formembrane proteins. Activation and signalling of cell surface proteinsare often involved in disease processes including malignanttransformation. Identification of selective target membrane proteins canbe used in the identification of biomarkers for early detection andprognosis of cancer but can also boost the discovery of new therapies.Moreover, mass spectrometry analysis has generated huge catalogues ofpossible bioactive peptides by the so-called peptidomics approaches. Formany of these peptides that produce a biological effect it is currentlynot clear what their targets are or which molecular mechanisms theyinitiate. Indeed, for such orphan peptides the cell surface receptors orother protein partners generally remain unknown.

Today, the detection and visualisation of proteins, and morespecifically cell surface proteins, is possible using a combination ofprimary and secondary antibodies. However, for the majority of cellsurface proteins, no antibodies are commercially available and, evenwhen available, the specificity (selectivity) and sensitivity (affinity)of commercially available polyclonal and monoclonal antibodies is oftenunsatisfactory for the detection of cell surface proteins. Furthermore,the use of antibodies is impossible for identifying unknown cell surfaceproteins.

Analysis of non-covalently bound cellular assemblies is, in particularfor cell surface receptors, difficult for several reasons. Theirhydrophobic nature and relatively low abundance precludes easy upscalingas they typically need to be in their natural environment to maintainbinding properties. Activation of receptors by their ligands at the cellsurface is based on the formation of a transient complex that, due todynamic turnover, can be rapidly internalized and degraded.Immunoaffinity-based techniques for isolating ligand-receptor complexesfrom cell lysates are therefore rarely successful and can only beapplied for high affinity interactions. Furthermore, interactomicstechniques such as the yeast two-hybrid screen are unsuitable for theidentification of extracellular protein-protein/peptide interactions.Elucidation of such interactions thus requires a technique compatiblewith living cells under physiological conditions.

In recent years, a whole range of bio-orthogonal chemistries wasdeveloped, allowing selective modification of biomolecules, in theirnatural environment. The introduced functional groups can react with apresented probe in an orthogonal way. Examples of such bio-orthogonalreactions include azide-alkyne cycloadditions, Staudinger ligation, andDiels Alder reactions. Despite the elegance and efficiency of thesemethods in labeling a wide variety of biomolecules, the need formodification of both binding partners with a specific unnatural reactivegroup represents a hurdle for general applicability.

Alternatively, photoaffinity crosslinking is based on the introductionof a photoreactive group which is able to form a crosslink with anunmodified natural binding partner upon activation with UV light. Thisrequirement for an activation step limits the applicability ofphoto-crosslinking in complex biological settings such as living cells.Benzophenones, aryl azides and diazirines are among the most widely usedgroups. Although previously applied in the characterization ofligand-receptor complexes, these chemistries bear several disadvantages.Phototoxicity needs to be strictly monitored. Furthermore, the formationof highly reactive intermediates reduces selectivity of crosslinking.Therefore, experiments are generally carried out with cell lysates or,when working with living cells, in cold buffers. Benzophenones aretypically bulky groups, which may negatively influence biologicalactivity of the used probes. Aryl azides are much smaller, but theshort-wave UV-light needed for their activation (<300 nm), is known tocause damage to the biological environment. Finally, diazirines are morestable, are excited at higher wavelengths, but their synthesis is quitetedious.

Recently, a crosslinking technology was developed based on a furanmoiety. Furan represents a latent reactive moiety that needs primaryoxidative activation to allow covalent bond formation with nucleophilicsites in its proximity. Furan, when incorporated in oligonucleotides,can be activated by oxidation using N-bromosuccinimide (NBS) (Halila etal., 2005, Chem. Commun. (Camb), 936-8) or singlet oxygen (Op de Beeckand Madder, 2012, J. Am. Chem. Soc., 134, 10737-40).

WO2012/085279 describes a method for crosslinking peptides comprising afuran moiety with second peptides. The method requires the addition ofan activation signal to oxidize the furan moiety. The example section ofWO2012/085279 illustrates that crosslinking furan-StrepTagII peptideswith streptavidin required the addition of NBS as an activation signalto oxidize the furan moiety.

Although furan is commercially available, its use in cell-based assayshas always been avoided in view of its toxicity and carcinogenicity. Inthe liver, cytochrome P450 catalyses oxidation of furan to a reactivealdehyde, which subsequently reacts with sulfhydryl and amine groups(Chen et al., 1997, Chem. Res. Toxicol., 10, 866-74).

In view of the above, there remains a need in the art to provide furtherand/or improved methods for detecting and/or identifying cell surfaceproteins or ligands specifically binding to cell surface proteins.

SUMMARY OF THE INVENTION

Prior art furan-based methods for crosslinking require an exogenousactivation signal (such as N-bromosuccinimide or light) for oxidativeactivation of the furan moiety. Unexpectedly, the present inventors havefound that the presence of such an activation signal is not necessary toobtain covalent binding between a cell surface protein and a ligand inliving cells. The example section herein illustrates that a cell surfaceprotein and a ligand, specifically binding to the cell surface protein,are covalently bound by contacting the living cells with the ligandcomprising a furan moiety.

Hence, a first aspect of the present invention relates to a method forcovalently binding a cell surface protein and a ligand, the ligand beingcapable of specifically binding to the cell surface protein, the methodconsisting essentially of contacting living cells expressing the cellsurface protein with the ligand comprising at least one furan moiety,thereby covalently binding the cell surface protein and the ligand.

The methods illustrating the principles of the present inventionadvantageously allow covalently binding a cell surface protein and aligand without the need for any exogenous (chemical or physical)activation. The present methods thus allow the formation of a covalentcell surface protein-ligand complex in situ under physiologicalconditions, e.g., using growth media. Advantageously, the presentmethods allow the covalent binding in diverse types of living cells suchas but not limited to human cells, mouse cells, cancerous cells, andhealthy cells. Furthermore, the present methods allow the covalentbinding of a cell surface protein and a ligand without loss ofefficiency or specificity compared to existing methods for crosslinkingperformed by the addition of an activation signal such as photoaffinitycrosslinking. The observed efficiency, selectivity, andproximity-induced specific covalent binding of the present methods incombination with their compatibility with physiological conditions andzero cell toxicity make the methods illustrating the present inventionsuperior to existing crosslinking techniques.

The present methods allow the identification of target cell surfaceproteins of biologically active orphan ligands, such as orphan peptidesor small molecules, without the requirement for exogenous activation.For instance, the present methods allow to screen peptide librariesgenerated by standard solid phase peptide synthesis and to identify thecell surface protein, e.g. via mass spectrometry-based sequencing, if acovalently bound complex is present. Next to identifying the cellsurface protein involved, the present methods allow to gain immediateinsight on the location of the peptide binding site. Alternatively, themethods illustrating the present invention allow in situ receptorlabeling, e.g. in diagnostics, as a cheap, reliable alternative toantibodies.

Hence, a further aspect relates to a cell-based assay for identifying,for a known ligand, a cell surface protein to which the ligand iscapable of specifically binding thereto, the ligand comprising at leastone furan moiety, the cell-based assay consisting essentially of:

-   -   contacting living cells with the ligand;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand; and    -   identifying the cell surface protein if the covalently bound        complex is present.

A further aspect relates to a cell-based assay for identifying, for aknown cell surface protein, a ligand specifically binding to the cellsurface protein, the cell-based assay consisting essentially of:

-   -   contacting living cells expressing the cell surface protein with        a ligand comprising at least one furan moiety;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand;    -   inferring from the finding that the covalently bound complex is        present that the ligand specifically binds the cell surface        protein.

Yet a further aspect relates to a cell-based assay for identifying abinding site of a cell surface protein and a peptide, the peptide beingcapable of specifically binding to the cell surface protein and thepeptide comprising at least one amino acid comprising a furan moiety,wherein said amino acid comprising a furan moiety is located at positionn of the peptide, the cell-based assay consisting essentially of:

-   (a) contacting living cells with the peptide;-   (b) determining the presence of a covalently bound complex of the    cell surface protein and the peptide;-   (c) identifying the amino acid comprising a furan moiety as a    binding site of the cell surface protein and the peptide if the    covalently bound complex is present;-   (d) optionally repeating steps (a) to (c) with peptides comprising a    furan moiety, wherein the amino acid comprising a furan moiety is    located at position n+p of the peptides comprising a furan moiety;    wherein position n may be any amino acid of the peptides comprising    a furan moiety, and wherein p is a positive or negative integer    (provided position n+p is located on the peptides comprising a furan    moiety).

The ensuing statements provide additional illustration of certainaspects and embodiments that have been disclosed in accordance with thepresent invention:

-   1. A method for covalently binding a cell surface protein and a    ligand, the ligand being capable of specifically binding to the cell    surface protein, the method consisting essentially of contacting    living cells expressing the cell surface protein with the ligand    comprising at least one furan moiety, thereby covalently binding the    cell surface protein and the ligand.-   2. The method according to statement 1, wherein the cell surface    protein is selected from the group consisting of a cell surface    receptor, a cell adhesion molecule, and a cell surface protease.-   3. The method according to statement 1 or 2, wherein the cell    surface protein is a cell surface receptor selected from the group    consisting of a G-protein coupled receptor (GPCR), an immune    receptor, an ion channel-linked receptor, and an enzyme-linked    receptor, preferably wherein the cell surface protein is a GPCR.-   4. The method according to any one of statements 1 to 3, wherein the    ligand is a peptide, a nucleoside, a nucleic acid, a lipid, a    polysaccharide, a small molecule, or a combination thereof,    preferably wherein the ligand is a peptide.-   5. The method according to any one of statements 1 to 4, wherein the    cell surface protein is a GPCR and the ligand is a peptide.-   6. The method according to any one of statements 1 to 5, wherein the    furan moiety of the ligand is oxidized by endogenous activation.-   7. The method according to statement 6, wherein the endogenous    activation occurs at the extracellular space of the cell membrane.-   8. The method according to any one of statements 1 to 7, wherein the    cell surface protein comprises at least one amine group, hydroxyl    group, sulfhydryl group, imidazole group and/or indole group.-   9. The method according to any one of statements 6 to 8, wherein the    oxidized furan moiety of the ligand reacts with the amine group,    hydroxyl group, sulfhydryl group, imidazole group and/or indole    group of the cell surface protein.-   10. The method according to any one of statements 1 to 9, wherein    the method is performed under physiological conditions.-   11. The method according to any one of statements 1 to 10, wherein    the living cells are normal cells.-   12. A method for detecting a cell surface protein covalently bound    to a ligand, the ligand being capable of specifically binding to the    cell surface protein, the method consisting essentially of:    -   performing the method as defined in any one of statements 1 to        11, and    -   detecting the cell surface protein covalently bound to the        ligand, preferably by flow cytometry, microscopy,        gel-electrophoresis, Western blot, immunoassays, mass        spectrometry, or a combination thereof.-   13. A cell-based assay for identifying, for a known ligand, a cell    surface protein to which the ligand is capable of specifically    binding thereto, the ligand comprising at least one furan moiety,    the cell-based assay consisting essentially of:    -   contacting living cells with the ligand;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand; and    -   identifying the cell surface protein if the covalently bound        complex is present.-   14. A cell-based assay for identifying, for a known cell surface    protein, a ligand specifically binding to the cell surface protein,    the cell-based assay consisting essentially of:    -   contacting living cells expressing the cell surface protein with        a ligand comprising at least one furan moiety;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand; and    -   inferring from the finding that the covalently bound complex is        present that the ligand specifically binds the cell surface        protein.-   15. A cell-based assay for identifying a binding site of a cell    surface protein and a peptide, the peptide being capable of    specifically binding to the cell surface protein and the peptide    comprising at least one amino acid comprising a furan moiety,    wherein said amino acid comprising a furan moiety is located at    position n of the peptide, the cell-based assay consisting    essentially of:-   (a) contacting living cells with the peptide;-   (b) determining the presence of a covalently bound complex of the    cell surface protein and the peptide;-   (c) identifying the amino acid comprising a furan moiety as a    binding site of the cell surface protein and the peptide if the    covalently bound complex is present;-   (d) optionally repeating steps (a) to (c) with peptides comprising a    furan moiety, wherein the amino acid comprising a furan moiety is    located at position n+p of the peptides comprising a furan moiety;    wherein position n may be any amino acid of the peptides comprising    a furan moiety, and wherein p is a positive or negative integer    (provided position n+p is located on the peptides comprising a furan    moiety).-   16. The cell-based assay according to any one of statements 13 to    15, wherein    -   the cell surface protein is selected from the group consisting        of a cell surface receptor, a cell adhesion molecule, and a cell        surface protease;    -   the cell surface protein is a cell surface receptor selected        from the group consisting of a G-protein coupled receptor        (GPCR), an immune receptor, an ion channel-linked receptor, and        an enzyme-linked receptor, preferably wherein the cell surface        protein is a GPCR;    -   the ligand is a peptide, a nucleoside, a nucleic acid, a lipid,        a polysaccharide, a small molecule, or a combination thereof,        preferably wherein the ligand is a peptide;    -   the cell surface protein is a GPCR and the ligand is a peptide;    -   the cell-based assay is performed under physiological        conditions; and/or    -   the living cells are normal cells.-   17. A method for covalently binding a cell surface protein and a    ligand, the ligand being capable of specifically binding to the cell    surface protein, the method comprising or consisting essentially of    contacting living cells expressing the cell surface protein with the    ligand comprising at least one furan moiety without the addition of    an exogenous activation signal, thereby covalently binding the cell    surface protein and the ligand.-   18. The method according to statement 17, wherein the cell surface    protein is selected from the group consisting of a cell surface    receptor, a cell adhesion molecule, and a cell surface protease.-   19. The method according to statement 17 or 18, wherein the cell    surface protein is a cell surface receptor selected from the group    consisting of a G-protein coupled receptor (GPCR), an immune    receptor, an ion channel-linked receptor, and an enzyme-linked    receptor, preferably wherein the cell surface protein is a GPCR.-   20. The method according to any one of statements 17 to 19, wherein    the ligand is a peptide, a nucleoside, a nucleic acid, a lipid, a    polysaccharide, a small molecule, or a combination thereof,    preferably wherein the ligand is a peptide.-   21. The method according to any one of statements 17 to 20, wherein    the cell surface protein is a GPCR and the ligand is a peptide.-   22. The method according to any one of statements 71 to 21, wherein    the furan moiety of the ligand is oxidized by endogenous activation.-   23. The method according to statement 22, wherein the endogenous    activation occurs at the extracellular space of the cell membrane.-   24. The method according to any one of statements 17 to 23, wherein    the cell surface protein comprises at least one amine group,    hydroxyl group, sulfhydryl group, imidazole group and/or indole    group.-   25. The method according to any one of statements 22 to 24, wherein    the oxidized furan moiety of the ligand reacts with the amine group,    hydroxyl group, sulfhydryl group, imidazole group and/or indole    group of the cell surface protein.-   26. The method according to any one of statements 17 to 25, wherein    the method is performed under physiological conditions.-   27. The method according to any one of statements 17 to 16, wherein    the living cells are normal cells.-   28. A method for detecting a cell surface protein covalently bound    to a ligand, the ligand being capable of specifically binding to the    cell surface protein, the method consisting essentially of:    -   performing the method as defined in any one of statements 17 to        27, and    -   detecting the cell surface protein covalently bound to the        ligand, preferably by flow cytometry, microscopy,        gel-electrophoresis, Western blot, immunoassays, mass        spectrometry, or a combination thereof.-   29. A cell-based assay for identifying, for a known ligand, a cell    surface protein to which the ligand is capable of specifically    binding thereto, the ligand comprising at least one furan moiety,    the cell-based assay comprising or consisting essentially of:    -   contacting living cells with the ligand without the addition of        an exogenous activation signal;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand; and    -   identifying the cell surface protein if the covalently bound        complex is present.-   30. A cell-based assay for identifying, for a known cell surface    protein, a ligand specifically binding to the cell surface protein,    the cell-based assay comprising or consisting essentially of:    -   contacting living cells expressing the cell surface protein with        a ligand comprising at least one furan moiety without the        addition of an exogenous activation signal;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand; and    -   inferring from the finding that the covalently bound complex is        present that the ligand specifically binds the cell surface        protein.-   31. A cell-based assay for identifying a binding site of a cell    surface protein and a peptide, the peptide being capable of    specifically binding to the cell surface protein and the peptide    comprising at least one amino acid comprising a furan moiety,    wherein said amino acid comprising a furan moiety is located at    position n of the peptide, the cell-based assay comprising or    consisting essentially of:    -   (a) contacting living cells with the peptide without the        addition of an exogenous activation signal;    -   (b) determining the presence of a covalently bound complex of        the cell surface protein and the peptide;    -   (c) identifying the amino acid comprising a furan moiety as a        binding site of the cell surface protein and the peptide if the        covalently bound complex is present;    -   (d) optionally repeating steps (a) to (c) with peptides        comprising a furan moiety, wherein the amino acid comprising a        furan moiety is located at position n+p of the peptides        comprising a furan moiety;    -    wherein position n may be any amino acid of the peptides        comprising a furan moiety, and wherein p is a positive or        negative integer (provided position n+p is located on the        peptides comprising a furan moiety).-   32. The cell-based assay according to any one of statements 29 to    31, wherein    -   the cell surface protein is selected from the group consisting        of a cell surface receptor, a cell adhesion molecule, and a cell        surface protease;    -   the cell surface protein is a cell surface receptor selected        from the group consisting of a G-protein coupled receptor        (GPCR), an immune receptor, an ion channel-linked receptor, and        an enzyme-linked receptor, preferably wherein the cell surface        protein is a GPCR;    -   the ligand is a peptide, a nucleoside, a nucleic acid, a lipid,        a polysaccharide, a small molecule, or a combination thereof,        preferably wherein the ligand is a peptide;    -   the cell surface protein is a GPCR and the ligand is a peptide;    -   the cell-based assay is performed under physiological        conditions; and/or    -   the living cells are normal cells.

In all of the above mentioned statements 1 to 16, the term “consistingessentially of” implies that no exogenous activation signal is added inthe methods or cell-based assays. This means that in the methods orcell-based assays of any one of the above aspects, the covalent bindingbetween the protein component and its ligand comprising a furan moietyis effectuated without the addition of an exogenous activation signal.

The present invention will now be further described. In the followingpassages, different aspects of the invention are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular, any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents a schematic overview illustrating the synthesis of afuran amino acid from a commercially available furyl amine derivativeand a glutamic acid derivative. (1): amide bond formation; (2): acidtreatment.

FIG. 2 represents a schematic overview illustrating the synthesis of afuran amino acid from a commercially available furyl amine derivativeand an aspartic acid derivative. (1): amide bond formation; (2): acidtreatment.

FIG. 3A and FIG. 3B represent a schematic overview illustrating thesynthesis of a furan amino acid from a lysine derivative and acommercially available furyl isocyanate derivative or a furyl carboxylicacid derivative, respectively.

FIG. 4 represents an overview illustrating the sequence of kisspeptin 10(compound 1) and analogues thereof (compounds 2 to 7). Except forcompound 1, the peptides were labeled at the N-terminus using eitherbiotin (2-5) or biotin-dPEG(4) (6 and 7). In peptides 2, 4, 6, 7furylalanine (FUA) substitutes the amino acid at position 3 (in 2, 6,and 7) or position 1 (in 4). Peptides 3 and 5 containbenzoylphenylalanine (BPA).

FIG. 5 represents a photograph of a Western blot illustrating thedifferent forms of receptor GPR54 in MDA-MB-231 cells. Western Blot ofcell lysate of MDA-MB-231 cells using an antibody against GPR54(arrowheads) and biotin-responsive streptavidin (diamonds). ReceptorGPR54 is present as three subspecies of 37 kDa, 54 kDa, and 72 kDa(arrowheads). These subspecies represent different states of maturationvia glycosylation and the 72 kDa is the receptor form presented at thecell surface. The signals from endogenous biotinylated proteins areshown (diamonds) as reference for the background signal in all biotinprobing results. The MW of the shown marker proteins (M) is indicated.

FIG. 6 represents photographs of a Western blot illustrating cellsurface receptor-ligand crosslink formation on living cells using acomparative method for crosslinking (using NBS as an activation signal).Western blot of cell lysates prepared after crosslinking furan modifiedkisspeptin peptides on MDA-MB-231 cells. FIG. 6A represents a photographof the Western blot using an antibody against GPR54. FIG. 6B representsa photograph of the same Western blot using biotin-responsivestreptavidin.

FIG. 7 represents a photograph of a Western blot illustrating cellsurface receptor-ligand crosslink formation on living cells usingcomparative methods for crosslinking (using NBS as an activation signalor using photo-crosslinking). Western blot of cell lysates preparedafter crosslinking furan-modified kisspeptin peptides on MDA-MB-231cells. Biotinylated proteins are visualized. Blank shows the backgroundsignals from endogenously biotinylated carboxylases (75 and 130 kDa,diamonds). The position of the crosslinked biotinylated cell surfacereceptor-ligand complex is indicated by an arrow.

FIG. 8 represents a photograph of a Western blot illustrating covalentbinding of a cell surface receptor and a ligand on living cells using amethod according to an embodiment of the present invention (lane 2) anda comparative method (lane 1, with addition of NB S). Western blot ofcell lysates prepared after covalently binding furan modified kisspeptinpeptides on MDA-MB-231 cells in growth medium comprising 10% serum. Onlythe signal for biotin is shown. Blank shows the background signals fromendogenously biotinylated carboxylases (75 and 130 kDa, diamonds). Theposition of the crosslinked biotinylated cell surface receptor-ligandcomplex is indicated by an arrow.

FIG. 9 represents photographs of a Western blot illustrating covalentbinding of a cell surface receptor and a ligand on living cells using amethod according to embodiments of the present invention. The effect ofthe presence of a PEG-linker between the N-terminus and biotin-moiety ofthe ligand on the efficiency of covalent binding is shown. FIG. 9Arepresents a photograph of the Western blot using an antibody againstGPR54. FIG. 9B represents a photograph of the same Western blot usingbiotin-responsive streptavidin. M: marker proteins.

FIG. 10 represents photographs of a Western blot illustrating covalentcell surface receptor-ligand complex formation on living cells using amethod according to an embodiment of the present invention. Western blotof cell lysates prepared after covalently binding furan modifiedkisspeptin peptides on MDA-MB-231 cells in growth medium comprising 10%serum. FIG. 10A represents a photograph of the Western blot using anantibody against GPR54. FIG. 10B represents a photograph of the sameWestern blot using biotin-responsive streptavidin. M: marker proteins.

FIG. 11 represents photographs of a Western blot illustrating thecovalent binding of furan modified kisspeptin and cell surface receptorGPR54 on NIH 3T3 cell line according to an embodiment of the presentmethod. Western blot of cell lysates prepared after covalently bindingfuran-modified kisspeptin peptide 6. FIG. 11A represents a photograph ofthe Western blot using an antibody against GPR54. FIG. 11B represents aphotograph of the same Western blot using biotin-responsivestreptavidin. Arrow: cell surface receptor-ligand complex (about 72kDa). Diamonds: biotinylated carboxylases (75 and 130 kDa); arrowhead:receptor GPR54. M: marker proteins.

FIG. 12 represents photographs of a Western blot illustrating thecovalent binding of furan modified kisspeptin and cell surface receptorGPR54 on HeLa cell line according to an embodiment of the presentmethod. Western blot of cell lysates prepared after covalently bindingfuran-modified kisspeptin peptide 6. FIG. 12A represents a photograph ofthe Western blot using an antibody against GPR54. FIG. 12B represents aphotograph of the same Western blot using biotin-responsivestreptavidin. Arrow: cell surface receptor-ligand complex (about 72kDa). Diamonds: biotinylated carboxylases (75 and 130 kDa); arrowhead:receptor GPR54. M: marker proteins.

FIG. 13 represents photographs of a Western blot illustrating thecovalent binding of furan modified kisspeptin and cell surface receptorGPR54 on MCF-7 cell line according to an embodiment of the presentmethod. Western blot of cell lysates prepared after covalently bindingfuran-modified kisspeptin peptide 6. FIG. 13A represents a photograph ofthe Western blot using an antibody against GPR54. FIG. 13B represents aphotograph of the same Western blot using biotin-responsivestreptavidin. Arrow: cell surface receptor-ligand complex (about 72kDa). Diamonds: biotinylated carboxylases (75 and 130 kDa); arrowhead:receptor GPR54. M: marker proteins.

FIG. 14 represents photographs of a Western blot illustrating thecovalent binding of furan modified kisspeptin and cell surface receptorGPR54 on HEK-myc-KISS1R cell line according to an embodiment of thepresent method. Western blot of cell lysates prepared after covalentlybinding furan-modified kisspeptin peptide 6. FIG. 14A represents aphotograph of the Western blot using an antibody against GPR54. FIG. 14Brepresents a photograph of the same Western blot using biotin-responsivestreptavidin. Arrow: cell surface receptor-ligand complex (about 72kDa). Diamonds: biotinylated carboxylases (75 and 130 kDa); arrowhead:receptor GPR54. M: marker proteins.

FIG. 15 represents an overview illustrating the sequence, chemicalformula, exact mass, and molecular weight of opioid peptides andfuran-modified analogues thereof.

DETAILED DESCRIPTION OF THE INVENTION

Before the present method and products of the invention are described,it is to be understood that this invention is not limited to particularmethods, components, products or combinations described, as suchmethods, components, products and combinations may, of course, vary. Itis also to be understood that the terminology used herein is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. The terms also encompass“consisting of” and “consisting essentially of”, which enjoywell-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The term “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, is meant to encompass variations of +/−10% or less,preferably +/−5% or less, more preferably +/−1% or less, and still morepreferably +/−0.1% or less of and from the specified value, insofar suchvariations are appropriate to perform in the disclosed invention. It isto be understood that the value to which the modifier “about” or“approximately” refers is itself also specifically, and preferably,disclosed.

Whereas the terms “one or more” or “at least one”, such as one or moreor at least one member(s) of a group of members, is clear per se, bymeans of further exemplification, the term encompasses inter alia areference to any one of said members, or to any two or more of saidmembers, such as, e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of said members,and up to all said members.

All references cited in the present specification are herebyincorporated by reference in their entirety. In particular, theteachings of all references herein specifically referred to areincorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention.

In the following passages, different aspects of the invention aredefined in more detail. Each aspect so defined may be combined with anyother aspect or aspects unless clearly indicated to the contrary. Inparticular, any feature indicated as being preferred or advantageous maybe combined with any other feature or features indicated as beingpreferred or advantageous.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to a person skilled in the art from this disclosure, in one ormore embodiments. Furthermore, while some embodiments described hereininclude some but not other features included in other embodiments,combinations of features of different embodiments are meant to be withinthe scope of the invention, and form different embodiments, as would beunderstood by those in the art. For example, in the appended claims, anyof the claimed embodiments can be used in any combination.

The present inventors have found that the presence of an exogenousactivation signal is not necessary to oxidize the furan moiety andobtain covalent binding of a cell surface protein and a ligand on livingcells.

Hence, the present invention provides a method for covalently binding acell surface protein and a ligand, the ligand being capable ofspecifically binding to the cell surface protein, the method consistingessentially of or consisting of contacting living cells expressing thecell surface protein with the ligand comprising at least one furanmoiety, thereby covalently binding the cell surface protein and theligand. The methods as taught herein are preferably performed in vitro.

The term “in vitro” generally denotes outside, or external to, animal orhuman body. The term “ex vivo” typically refers to tissues or cellsremoved from an animal or human body and maintained or propagatedoutside the body, e.g., in a culture vessel. The term “in vitro” as usedherein should be understood to include “ex vivo”. The term “in vivo”generally denotes inside, on, or internal to, animal or human body.

The present invention provides methods for covalently binding a cellsurface protein as described herein and a ligand as described herein.

The terms “covalently binding”, “covalent coupling”, or “crosslinking”may be used interchangeably herein and refer to coupling a cell surfaceprotein and a ligand with a covalent bond. The covalent bond renders thebinding permanent as opposed to a transient binding.

The method advantageously allows site-selective coupling of a ligand toits cell surface receptor. The term “site-selective coupling”, asopposed to non-selective or non-specific coupling, refers to thecoupling of one or more determined monomers present or introduced in oneor more determined positions of a ligand (e.g., one or more furan aminoacids present or introduced in one or more determined positions of thefuran-peptide) to a cell surface receptor.

Accordingly, in an embodiment, the invention relates to a method for thesite-selective covalent binding of a cell surface protein and a ligand,the ligand being capable of specifically binding to the cell surfaceprotein, the method consisting essentially of contacting the livingcells expressing the cell surface protein with the ligand comprising atleast one furan moiety.

Cell Surface Protein

The term “cell surface protein” as used herein refers to any proteinpresent on the extracellular surface of a cell.

The terms “protein”, “polypeptide”, or “peptide” can be usedinterchangeably and relate to any natural or recombinant moleculecomprising amino acids joined together by peptide bonds between adjacentamino acid residues. A “peptide bond”, “peptide link” or “amide bond” isa covalent bond formed between two amino acids when the carboxyl groupof one amino acid reacts with the amino group of the other amino acid,thereby releasing a molecule of water. The terms “amino acid” and “aminoacid residue” may be used interchangeably herein.

The recitation “protein being present on the extracellular surface of acell” encompasses that at least part of the protein is exposed to theextracellular space (or compartment) of the cell.

The “extracellular space of the cell” refers to the space (orcompartment) at the extracellular or external side of the plasmamembrane (i.e., cell membrane).

The number of amino acids of the cell surface protein is not limiting.In certain embodiments, the cell surface protein as taught herein maycontain at least 20 amino acids. In certain embodiments, the cellsurface protein as taught herein may contain from 20 to 20000 aminoacids. In certain embodiments, the cell surface protein as taught hereinmay contain from 50 to 5000 amino acids, for example, the cell surfaceprotein as taught herein may contain from 50 to 100 amino acids, or from100 to 1000 amino acids, or from 1000 to 5000 amino acids. In certainembodiments, the cell surface protein as taught herein may contain from5000 to 20000 amino acids, for example, the cell surface protein astaught herein may contain from 5000 to 10000 amino acids, or from 10000to 20000 amino acids. For instance, the cell surface proteins as taughtherein contain at least 20, at least 50, at least 100, at least 200, atleast 300, at least 400, at least 500, at least 600, at least 700, atleast 800, at least 900, at least 1000, at least 1500, at least 2000, atleast 2500, at least 3000, at least 3500, at least 4000, or at least4500 amino acids.

In certain embodiments, the cell surface protein may be an integralmembrane protein, i.e., a protein which is permanently embedded in thecell membrane. Particularly, in certain embodiments, the cell surfaceprotein may be an integral polytopic protein or transmembrane protein(i.e., an integral membrane protein that spans across the cell membraneat least once) or an integral monotopic protein (i.e., an integralmembrane protein that is exposed on only one side (the extracellularside) of the cell membrane and does not span the whole way across thecell membrane).

In certain embodiments, the cell surface protein may be a peripheralmembrane protein, i.e., a protein which is temporarily (and hencereversibly) attached either to the lipid bilayer of the cell membrane orto integral membrane proteins by a combination of hydrophobic,electrostatic, and other non-covalent interactions.

In certain embodiments, the cell surface protein may be a lipid-anchoredprotein (also known as lipid-linked protein), i.e., a protein which iscovalently linked to a lipid, a glycolipid, or aglycosylphosphatidylinositol lipid that has its fatty acids inserted inthe lipid bilayer of the cell membrane.

In certain embodiments, the cell surface protein may be modified, forinstance by post-translational modification. For instance, the cellsurface protein may be phosphorylated, tyrosine sulfated, and/orglycosylated. Post-translational modification of proteins can beexperimentally detected by a variety of techniques, including massspectrometry and Western blotting.

In certain embodiments, the cell surface protein may be an endogenousprotein. This advantageously allows studying endogenous cell surfaceproteins, e.g., by visualizing the localization, internalization,trafficking and diffusion characteristics of endogenous cell surfaceproteins in the cell membrane.

The term “endogenous protein” refers to a protein resulting fromexpression of a nucleic acid, such as DNA, naturally found in the genomeof the cell in which the protein is expressed.

By “nucleic acid” is meant oligomers and polymers of any length composedessentially of nucleotides, e.g., deoxyribonucleotides and/orribonucleotides. Nucleic acids can comprise purine and/or pyrimidinebases and/or other natural (e.g., xanthine, inosine, hypoxanthine),chemically or biochemically modified (e.g., methylated), non-natural, orderivative nucleotide bases. The backbone of nucleic acids can comprisesugars and phosphate groups, as can typically be found in RNA or DNA,and/or one or more modified or substituted sugars and/or one or moremodified or substituted phosphate groups. Modifications of phosphategroups or sugars may be introduced to improve stability, resistance toenzymatic degradation, or some other useful property. A “nucleic acid”can be for example double-stranded, partly double stranded, orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear. The term “nucleic acid” as used herein preferablyencompasses DNA and RNA, specifically including RNA, genomic RNA, cDNA,DNA, provirus, pre-mRNA and mRNA.

In certain embodiments, the cell surface protein may be an exogenousprotein.

The term “exogenous protein” refers to a protein resulting fromexpression of a nucleic acid, such as DNA, not naturally found in thegenome of but introduced into the cell in which the protein isexpressed. The nucleic acid, such as DNA, can be transiently introducedin the cell in which the protein is expressed or can be stablyintroduced in the genome of the cell in which the protein is expressed.The nucleic acid, such as DNA, can be introduced in the cell in whichthe protein is expressed via methods known in the art such astransfection or viral infection (transduction). The nucleic acid, suchas DNA, can be a derived from the genome of a different organism. Thenucleic acid, such as DNA, can be wild type nucleic acid or recombinantnucleic acid.

In certain embodiments, the cell surface protein may be a wild-typeprotein. In certain embodiments, the cell surface protein may be anative protein.

As used herein, the term “wild-type” as applied to a nucleic acid orprotein refers to a nucleic acid or a protein that occurs in, or isproduced by, a biological organism as that biological organism exists innature. The term “wild-type” may to some extent be synonymous with“native”, the latter encompassing nucleic acids or proteins having anative sequence, i.e., ones of which the primary sequence is the same asthat of the nucleic acids or proteins found in or derived from nature. Askilled person understands that native sequences may differ between orwithin different individuals of the same species due to normal geneticdiversity (variation) within a given species. Also, native sequences maydiffer between or within different individuals of the same species dueto post-transcriptional or post-translational modifications. Any suchvariants or isoforms of nucleic acids or polypeptides are encompassedherein as being “native”. Accordingly, all sequences of nucleic acids orproteins found in or derived from nature are considered “native”.

In certain embodiments, the cell surface protein may be a recombinantprotein.

The term “recombinant” is generally used to indicate that the material(e.g., a nucleic acid, a genetic construct or a protein) has beenaltered by technical means (i.e., non-naturally) through humanintervention. The term “recombinant nucleic acid” commonly refers tonucleic acids comprised of segments joined together using recombinantDNA technology. The term may denote material (e.g., a nucleic acid, agenetic construct or a protein) that has been altered by technical meansof mutagenesis. As used herein the term “recombinant protein” refers toa protein that can result from the expression of recombinant nucleicacid such as recombinant DNA.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be selected from the groupconsisting of a cell surface receptor, a cell adhesion molecule, and acell surface protease.

The terms “cell surface receptor”, “membrane receptor”, or“transmembrane receptor” can be used interchangeably herein and refer toa receptor present on the extracellular surface of a cell.

The term “receptor” generally refers to a protein that is capable ofacting in cell signaling by receiving (specifically binding to) anextracellular agent (referred to herein as the “ligand”).

The term “cell-adhesion molecule (CAM)” refers to a protein located onthe surface of a cell and involved in binding with other cells or withthe extracellular matrix (ECM), i.e., involved in cell adhesion.

CAMs are typically transmembrane receptors composed of three parts: anintracellular domain that interacts with the cytoskeleton, atransmembrane domain, and an extracellular domain that interacts eitherwith other CAMs of the same kind (homophilic binding) or with other CAMsor the extracellular matrix (heterophilic binding).

In certain embodiments, the CAM is a calcium-independent CAM. In certainembodiments, the CAM is a calcium-independent CAM selected from animmunoglobulin superfamily CAM (IgSF CAM) or a lymphocyte homingreceptor. Examples of lymphocyte homing receptors or addressins are CD34and GLYCAM-1.

In certain embodiments, the CAM is a calcium-dependent CAM. In certainembodiments, the CAM is a calcium-dependent CAM selected from the groupconsisting of integrins, cadherins, and selectins.

In certain embodiments, the CAM is a cell-surface receptor.

The term “cell surface protease” or “membrane anchored protease” refersto a protein located on the surface of a cell and comprising a proteasedomain.

Non-limiting examples of membrane anchored proteases are A DisintegrinAnd Metalloproteinase domain-containing proteins (ADAM) or sheddase, andmembrane-type matrix metalloproteinases (MT-MMPs).

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be a cell surface receptor selectedfrom the group consisting of a G-protein coupled receptor (GPCR), animmune receptor, an ion channel-linked receptor, and an enzyme-linkedreceptor.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be a GPCR.

The terms “G protein-coupled receptor (GPCR)”, “seven-transmembranedomain receptor”, “7TM receptor”, “heptahelical receptor”, “serpentinereceptor”, or “G protein-linked receptor (GPLR)” refer to receptors thatpossess seven transmembrane helices.

Upon ligand binding, GPCRs activate a G protein located on theintracellular side. G proteins are trimeric proteins. The 3 subunits ofa G protein are called α, β, and γ. When a ligand binds to the GPCR itcauses a conformational change in the GPCR, which allows it to act as aguanine nucleotide exchange factor. The GPCR can then activate anassociated G protein by exchanging its bound GDP for a GTP. The Gprotein's a subunit, together with the bound GTP, can then dissociatefrom the β and γ subunits to further affect intracellular signalingproteins or target functional proteins directly depending on the asubunit type.

G protein-coupled receptors are found only in eukaryotes including yeastand animals.

Although numerous classification schemes have been proposed, thesuperfamily was classically divided into three main classes (A, B and C)with no detectable shared sequence homology between classes. Morerecently, an alternative classification system called GRAFS (Glutamate,Rhodopsin, Adhesion, Frizzled/Taste2, Secretin) has been proposed. Bythis system, GPCRs can be grouped into 6 classes based on sequencesimilarity (homology) and functional similarity: Class A (or 1)(Rhodopsin-like); Class B (or 2) (Secretin receptor family); Class C (or3) (Metabotropic glutamate/pheromone); Class D (or 4) (Fungal matingpheromone receptors); Class E (or 5) (Cyclic AMP receptors); or Class F(or 6) (Frizzled/Smoothened).

In certain embodiments of the methods or cell-based assays as taughtherein, the GPCR may be KiSS1-derived peptide receptor (GPR54), C—X—Cchemokine receptor type 4 (CXCR4), or C—C chemokine receptor type 5(CCR5).

In certain embodiments, the cell surface protein may be GPR54 and theligand may be kisspeptin-10 peptide.

GPR54 (also known as Kisspeptin receptor) is a G protein-coupledreceptor which binds the peptide hormone kisspeptin (metastin).

In certain embodiments, the cell surface protein may be CXCR4 and theligand may be CVX15.

CXCR4 (also known as CXCR-4 or fusin or CD184) is an alpha-chemokinereceptor for stromal-derived-factor-1 (SDF-1, also called CXCL12). CVX15is a peptide antagonist of the G-protein coupled receptor CXCR4.

In certain embodiments, the cell surface protein may be CCR5 and theligand may be Maraviroc.

CCR5 (also known as CD195) is a protein on the surface of white bloodcells that is involved in the immune system as it acts as a receptor forchemokines. Maraviroc is a small molecule antagonist of CCR5.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be an immune receptor.

The terms “immune receptor” and “immunologic receptor” as used hereinrefer to a cell surface receptor which binds to an extracellular agent(for example, a cytokine) and causes a response in the immune system.

In certain embodiments, the immune receptors may be pattern recognitionreceptors (PRRs) such as Toll-like receptors (TLRs) or NOD-likereceptors (NLRs); killer activated receptors (KARs); killer inhibitorreceptors (KIRs); complement receptors; Fc receptors; B cell receptors;T cell receptors; or cytokine receptors.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be an ion channel-linked receptor.

The terms “ion channel-linked receptors” or “ligand-gated ion channels(LGICs)” refer to receptors comprising a transmembrane domain includingan ion pore, and an extracellular domain including a ligand bindinglocation (an allosteric binding site). Ion channel-linked receptorsallow ions (such as Na⁺, K⁺, Ca²⁺, or Cl⁻) to pass through the cellmembrane in response to the binding of a ligand such as aneurotransmitter.

The ion channel-linked receptors constitute a large family of multi-passtransmembrane proteins. LGICs are classified into three families whichlack evolutionary relationship: Cys-loop receptors, Ionotropic glutamatereceptors and ATP-gated channels.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be an enzyme-linked receptor.

The terms “enzyme-linked receptors” or “catalytic receptors” refer toreceptors comprising an extracellular ligand-binding domain, atransmembrane helix, and an intracellular catalytic domain.

Typically, the binding of an extracellular agent to an enzyme-linkedreceptor causes direct or indirect enzymatic activity on theintracellular side.

Enzyme-linked receptors are typically single-pass transmembranereceptors, with the enzymatic component of the receptor keptintracellular. There are six known types of enzyme-linked receptors:Receptor tyrosine kinases; Tyrosine kinase associated receptors;Receptor-like tyrosine phosphatases; Receptor serine/threonine kinases;Receptor Guanylyl cyclases; and Histidine kinase associated receptors.

Ligand

The term “ligand” refers to any agent capable of comprising at least onefuran moiety. The nature of the ligand is not limited as long as theligand is capable of comprising at least one furan moiety. In certainembodiments, the ligand may be a natural ligand such as aneurotransmitter (e.g., serotonin, dopamine, gamma-aminobutyric acid(GABA), or glutamate). In certain embodiments, the ligand may be asynthetic or artificial ligand such as a drug.

The term “furan moiety”, “furan”, “furyl” or “furyl-moiety” as usedherein relates to a heterocyclic organic compound or functional groupconsisting of a five-membered aromatic ring with four carbon atoms andone oxygen atom.

In certain embodiments, the furan moiety may be bound to the ligand withone or two carbon atoms of the furan moiety. In certain embodiments, thefuran moiety may be bound to the ligand with two carbon atoms of thefuran moiety. For instance, the furan moiety may be bound to the ligandwith the carbon atoms at position 2 and 3, or with the carbon atoms atposition 2 and 4, or with the carbon atoms at position 2 and 5, or withthe carbon atoms at position 3 and 4 of the furan moiety.

In certain preferred embodiments, the furan moiety may be bound to theligand with one carbon atom of the furan moiety. In certain preferredembodiments, the furan moiety may be bound to the ligand with one carbonatom at position 2 of the furan moiety. In certain preferredembodiments, the furan moiety may be bound to the ligand with one carbonatom at position 3 of the furan moiety. Hence, in certain embodiments,the ligand may be a ligand comprising at least one furan moiety ofFormula Ia or Formula Ib. In certain embodiments, the ligand may be aligand comprising at least one furan moiety of Formula Ia. Such ligandsadvantageously allow efficient crosslinking of the ligand with the cellsurface protein.

In certain embodiments, the ligands as taught herein may comprise atleast one furan moiety. In certain embodiments, the ligands as taughtherein may comprise more than one, such as, for instance, 2, 3, 4, 5, 6,7, 8, 9, or 10 furan moieties. In certain embodiments, the ligands maycomprise only one furan moiety. The furan moieties as taught herein canbe located on any position in the ligand.

In certain embodiments, the ligands as taught herein may be provided insolution. The ligands as taught herein may be provided in a solventwherein the ligands can be dissolved. In certain embodiments, thesolvent is a polar protic solvent (such as water, methanol, or ethanol),a polar aprotic solvent (such as DMSO, dimethylformamide (DMF),acetonitrile, or tetrahydrofuran (THF)), or a non-polar solvent (such aschloroform or dichloromethane (DCM)). In certain embodiments, theligands as taught herein may be provided in a solvent comprising orconsisting of a polar protic solvent (such as water, methanol, orethanol), a polar aprotic solvent (such as DMSO, DMF, acetonitrile, orTHF), or an apolar solvent (such as chloroform or DCM).

In certain embodiments, the ligand may be capable of specificallybinding to the cell surface protein.

The terms “specifically binding” or “specific binding” as used hereinrefer to the ability of a ligand to preferentially bind to a particularcell surface protein that is present alongside different cell surfaceproteins. In certain embodiments, a specific binding interaction willdiscriminate between desirable (target) and undesirable (non-target)cell surface proteins, in some embodiments more than about 10-fold, morethan about 100-fold (e.g., more than about 1000- or 10,000-fold).

Accordingly, a ligand as taught herein is said to “specifically bind to”a particular cell surface protein when that ligand has affinity for,specificity for and/or is specifically directed to that cell surfaceprotein (or to at least one part or fragment thereof). The dissociationconstant (K_(d)) of the ligand as taught herein and the cell surfaceprotein as taught herein relates to the affinity between the ligand andthe cell surface protein.

The “specificity” of a ligand can be determined based on affinity forthe cell surface protein. In certain embodiments, a ligand is capable ofspecifically binding to a cell surface protein when the dissociationconstant of the ligand and the cell surface protein is at most 10 μM(i.e., 10⁻⁵ M). For instance, a ligand may be capable of specificallybinding to a cell surface protein when the dissociation constant of theligand and the cell surface protein is at most 1 μM (i.e., 10⁻⁶ M or 10³nM), at most 10² nM, at most 10 nM, at most 1 nM, at most 0.1 nM, atmost 0.01 nM, at most 1 pM, or at most 1 fM. Preferably, a ligand iscapable of specifically binding to a cell surface protein when thedissociation constant of the ligand and the cell surface protein is atmost 1 nM (i.e., 10⁻⁹ M), more preferably at most 1 pM (i.e., 10⁻¹² M).

A ligand as taught herein is said to be “specific for a first cellsurface protein as opposed to a second cell surface protein” when itbinds to the first cell surface protein with an affinity that is atleast 5 times, such as at least 10 times, such as at least 100 times,and preferably at least 1000 times higher than the affinity with whichthat ligand as taught herein binds to the second cell surface protein.Accordingly, in certain embodiments, when a ligand as taught herein issaid to be “specific for” a first cell surface protein as opposed to asecond cell surface protein, it may specifically bind to (as definedherein) the first cell surface protein but not to the second cellsurface protein.

In certain embodiments, the ligand as taught herein may comprise alabel.

The term “label” as used herein refers to any atom, molecule, moiety orbiomolecule that can be used to provide a detectable and preferablyquantifiable read-out or property, and that can be attached to or madepart of an entity of interest, such as the ligand as taught herein.Labels may be suitably detectable by mass spectrometric, spectroscopic,optical, colorimetric, magnetic, photochemical, biochemical,immunochemical or chemical means. Labels include without limitationdyes; radiolabels such as ³²P, ³³P, ³⁵S, ¹²⁵I, ¹³¹I; electron-densereagents; enzymes (e.g., horse-radish peroxidase (HRP) or alkalinephosphatase as commonly used in immunoassays); binding moieties such asbiotin-streptavidin; haptens such as digoxigenin; nanoparticles such asgold nanoparticles or quantum dots; luminogenic, phosphorescent orfluorogenic moieties; mass tags; and fluorescent agents, alone or incombination with moieties that can suppress or shift emission spectra byfluorescence resonance energy transfer (FRET).

In certain embodiments, the ligand as taught herein may comprise afluorophore, a non-fluorescent label, or a combination thereof.

In certain embodiments, the ligand as taught herein may comprise afluorophore. The fluorophore may advantageously serve as a marker (ordye, or tag, or reporter) for the cell surface protein to which theligand is bound. The fluorophore may allow to detect the cell surfaceprotein to which the ligand is bound in a variety of analytical methodsas defined herein such as fluorescent imaging, spectroscopy, andfluorescence-activated cell sorting (FACS).

The term “fluorophore”, “fluorescent agent”, or “fluorescent probe”generally refers to a chemical compound that can re-emit light uponlight excitation.

In certain embodiments, the fluorophore may comprise a moiety excitableby near infrared (NIR) light such as boron-dipyrromethene (BODIPY),rhodamine, cyanine, phthalocyanines, and squaraine. In certainembodiments, the fluorophore may comprise a fluorescein moiety, arhodamine moiety, a coumarin moiety, a cyanine moiety, BODIPY moiety,phthalocyanine moiety, or squaraine moiety.

In certain embodiments, the fluorophore may comprise or consist offluorescent nanoparticles such as quantum dots or nanocrystals.

Suitable non-limiting examples of commercially available fluorophoresare Alexa Fluor® Dyes (Thermo Fischer Scientific Inc., Massachusetts,USA), DyLight® Fluors (Thermo Fischer Scientific Inc., Massachusetts,USA), BODIPY Dyes (Thermo Fischer Scientific Inc., Massachusetts, USA),VivoTag Fluorochromes (PerkinElmer, Mass., USA), XenoLight CF™fluorescent labeling dyes (PerkinElmer, Mass., USA), Atto Dyes(Sigma-Aldrich, MO, USA), and Tracy Dyes (Sigma-Aldrich, MO, USA).

In certain embodiments, the ligand as taught herein may comprise anon-fluorescent label including but not limited to dyes; radiolabelssuch as ³²P, ³³P, ³⁵S, ¹²⁵I, ¹³¹I; electron-dense reagents; enzymes(e.g., horse-radish peroxidase (HRP) or alkaline phosphatase as commonlyused in immunoassays); biotin-streptavidin; haptens such as digoxigenin;and mass tags. The non-fluorescent label such as biotin may allowconjugation, isolation, and purification of the cell surface protein towhich the ligand is bound.

The label may be coupled to the ligand by methods known in the art. Forinstance, the label may be coupled to the ligand by N-terminalmodification during solid phase peptide synthesis (SPPS). Alternatively,introduction of an orthogonally protected Lysine derivative can befollowed by selective deprotection and labeling. Also, asite-specifically incorporated and unique cysteine residue can bemodified using fluorescent maleimides. Introduction of orthogonalfunctionalities, such as azide, alkyne and alkene, can be followed bylabeling through click chemistry approaches, such as azide/alkyne,tetrazine/alkene, and thiol/ene.

For example, the label may be a mass-altering label. Preferably, amass-altering label may involve the presence of a distinct stableisotope in one or more amino acids of a peptide vis-à-vis itscorresponding non-labelled peptide. Mass-labelled peptides areparticularly useful as positive controls, standards and calibrators inmass spectrometry applications. In particular, peptides including one ormore distinct isotopes are chemically alike, separatechromatographically and electrophoretically in the same manner and alsoionise and fragment in the same way. However, in a suitable massanalyser such peptides and optionally select fragmentation ions thereofwill display distinguishable m/z ratios and can thus be discriminated.Examples of pairs of distinguishable stable isotopes include H and D,¹²C and ¹³C, ¹⁴N and ¹⁵N or ¹⁶O and ¹⁸O. Usually, peptides and proteinsof biological samples may substantially only contain common isotopeshaving high prevalence in nature, such as for example H, ¹²C, ¹⁴N and¹⁶O. In such case, the mass-labelled peptide may be labelled with one ormore uncommon isotopes having low prevalence in nature, such as forinstance D, ¹³C, ¹⁵N and/or ¹⁸O. It is also conceivable that in caseswhere the peptides or proteins of a biological sample would include oneor more uncommon isotopes, the mass-labelled peptide may comprise therespective common isotope(s).

Isotopically-labelled synthetic peptides may be obtained inter alia bysynthesis or recombinant production of such peptides using one or moreisotopically-labelled amino acid substrates, or by chemically orenzymatically modifying unlabelled peptides to introduce thereto one ormore distinct isotopes. By means of example and not limitation,D-labelled peptides may be synthesised or produced by recombinantproduction in the presence of commercially available deuteratedL-methionine CH₃—S-CD₂CD₂-CH(NH₂)—COOH or deuterated arginineH₂NC(═NH)—NH—(CD₂)₃-CD(NH₂)—COOH. It shall be appreciated that any aminoacid of which deuterated or ¹⁵N- or ¹³C-containing forms exist may beconsidered for synthesis or recombinant production of labelled peptides.In another non-limiting example, a peptide may be treated with trypsinin H₂ ¹⁶O or H₂ ¹⁸O, leading to incorporation of two oxygens (¹⁶O or¹⁸O, respectively) at the COOH-termini of said peptide (e.g., US2006/105415).

In certain embodiments of the methods or cell-based assays as taughtherein, the ligand may be a peptide, a nucleoside, a nucleic acid, alipid, a polysaccharide, a small molecule, or a combination thereof.

In certain embodiments of the methods or cell-based assays as taughtherein, the ligand as taught herein may be a peptide.

Accordingly, in certain embodiments, the present invention relates to amethod for covalently binding a cell surface protein and a peptide, thepeptide being capable of specifically binding to the cell surfaceprotein, the method consisting essentially of contacting living cellsexpressing the cell surface protein with the peptide comprising at leastone furan moiety, thereby covalently binding the cell surface proteinand the peptide.

The term “furan-peptide” as used herein refers to any peptide comprisinga furan moiety. The methods or cell-based assays as taught herein maymake use of furan-peptides.

In certain embodiments, the furan-peptide is a peptide comprising afuran moiety of Formula Ia.

The furan-peptides as taught herein comprise at least one furan moiety.The furan-peptides may comprise more than one, such as, for instance, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 furanmoieties. In certain embodiments, the furan-peptide may comprise onlyone furan moiety. The furan moieties as taught herein can be located atany position in the furan-peptide, such as, for instance, N-terminally,C-terminally or internally. For instance, in case of more than one furanmoiety, a first furan moiety may be located C-terminally and a furtherfuran moiety may be located internally and/or N-terminally in afuran-peptide.

The furan-peptides as taught herein can be obtained by any suitablemethod known by the person skilled in the art.

In certain embodiments, the furan-peptides as taught herein can beobtained by incorporating at least one furan moiety during solid-phasepeptide synthesis (SPPS) of a peptide. Solid-phase peptide synthesis isa method that is widely used to chemically synthesize peptides (see,e.g., Merrifield, 1963, JACS, 85, 2149-2154) and can be adapted toproduce furan-peptides. This technique typically comprises two stages:the first stage of solid phase peptide synthesis (SPPS) includes theassembly of a peptide chain using protected amino acid derivatives on asolid support via repeated cycles of coupling-deprotection. The freeN-terminal amine of a solid-phase attached peptide can then be coupledto the C-terminal carboxyl of a single N-protected amino acid unit,e.g., a furan amino acid. This unit is then deprotected, revealing a newN-terminal amine to which a further amino acid may possibly be attached.While the peptide is being synthesized usually by stepwise methods, allsoluble reagents can be removed from the peptide-solid support matrix byfiltration and washed away at the end of each coupling step. In thesecond stage of SPPS, the peptide is cleaved from the support andside-chain protecting groups are removed to produce the peptide, e.g., afuran-peptide.

There are two major used forms of solid phase peptide synthesis: Fmoc(Carpino et al., 1972, J. Org. Chem., 37, 3404-3409), in which a baselabile alpha-amino protecting group is used, and t-Boc, in which an acidlabile protecting group is used. Each method involves different solidsupport resins and amino acid side chain protection and consequentcleavage/deprotection steps. For additional details regarding peptidesynthesis, see the following publications and references cited within:Crick et al., 1961, Nature, 192, 1227-32; Hofmann et al., 1966, JACS,88, 5914-9; Kaiser et al., 1989, Acc. Chem. Res., 22, 47-54; Nakatsukaet al., 1987, JACS, 109, 3808-10; Schnolzer et al., 1992, Science, 5054,221-5; Chaiken et al., 1981, CRC Crit. Rev. Biochem., 11, 255-301;Offord, 1987, Protein Eng, 1, 151-157; and Jackson et al., 1994,Science, 5183: 243-7; all of which are incorporated herein explicitly byreference.

In certain embodiments, the furan-peptides as taught herein can beobtained by incorporating at least one furan amino acid into a peptideduring protein translation in prokaryotes such as bacteria, e.g. E.coli, or in eukaryotes such as yeast or mammalian cells, as described byYoung and Schultz (2010, J. Biol. Chem., 285(15), 11039-44).

The genetic encoding of a furan amino acid in Escherichia coli and inhuman cells has been described by Schmidt et al. (Schmidt et al., 2013,Angew. Chem. Int. Ed., 52, 4690-4693; Schmidt et al., 2013, Angew.Chem., 125, 4788-4791; Schmidt et al., 2014, Chem Bio Chem.,15(12):1755-60).

The furan amino acid as taught herein can be any amino acid comprising afuran moiety, for example, the furan amino acid as taught herein may beselected from a furyl-glycine, furyl-alanine, furyl-valine,furyl-leucine, furyl-isoleucine, furyl-proline, furyl-tyrosine,furyl-tryptophan, furyl-phenylalanine, furyl-cysteine, furyl-methionine,furyl-serine, furyl-threonine, furyl-lysine, furyl-arginine,furyl-histidine, furyl-aspartic acid, furyl-glutamic acid,furyl-asparagine or furyl-glutamine. In certain embodiments, the furanamino acid as taught herein may be furyl-alanine, furyl-glycine, orfuryl-phenylalanine. Preferably, the furan amino acid as taught hereinmay be furylalanine, for example furyl-L-alanine or furyl-D-alanine,more preferably furyl-L-alanine. An important advantage of furyl-alanineis that it is commercially available. Moreover, furyl-alanine can beconsidered isosteric with histidine and iso-electronic with histidineand tyrosine. Consequently, no or minimal destabilization or alterationof the native protein structure is expected when incorporatingfurylalanine in a peptide.

The furan amino acid as taught herein can be an Fmoc-protected ortBoc-protected furan amino acid which allows easy incorporation intopeptides through solid-phase peptide synthesis. For example,Fmoc-protected furyl-alanine, which provides the required handle forsubsequent orthogonal labeling, is a commercially available amino acid.

The furan amino acid as taught herein may further be a furan amino acidas described by Schmidt et al. (2014, Chem Bio Chem., 15(12), 1755-60).

The furan amino acid as taught herein may further be obtained throughstandard organic synthesis using commercially available furanderivatives and commercially available amino acid derivatives.Commercially available furan derivatives used in the methods asdescribed herein comprise both 2- and 3-substituted furan derivatives.For instance, commercially available furan derivatives can be selectedfrom, but are not limited to compounds of Formula (IIIa), (IIIb),(IIIc), (IIId), (IIIe), (IIIf) or (IIIg), (IIIh), (IIIi), (IIIj), (IIIk)or stereoisomeric forms thereof.

Starting from commercially available furan derivatives, other furanderivatives are within reach through standard organic synthesis known tothe skilled person.

Commercially available amino acid derivatives used in the methods asdescribed herein can for example be selected from but are not limited tocompounds with Formula (IVa) or (IVb), or stereoisomeric forms thereof,

wherein n is an integer selected from 0, 1 or 2.

In an embodiment, the furan amino acids as taught herein are obtainedthrough amide bond formation between a furyl amine derivative and thecarboxyl group of a glutamic acid (Glu) derivative as depicted inFIG. 1. Furthermore, in an embodiment, the furan amino acids as taughtherein may be obtained through amide bond formation between a furylamine derivative and the carboxyl group of an aspartic acid (Asp)derivative as shown in FIG. 2. In an embodiment, the furan amino acidsas taught herein may further be obtained through amide bond formationbetween a furyl isocyanate derivative or a furyl carboxylic acidderivative and the amine group of Lys as shown in FIG. 3A and FIG. 3B,respectively.

In certain embodiments, the furan amino acid as taught herein can belocated in any position in the peptide. It will be understood by theskilled person, however, that sterical hindrance, e.g. of the furanamino acid, by other amino acids of the peptide should preferably beavoided. The furan amino acid as taught herein is preferably located ina position in the furan-peptide being accessible for binding to the cellsurface protein. The position of the furan amino acid or furan moiety astaught herein in the peptide is preferably chosen based on, e.g.,whether its position in a particular location would change theconformation, activity or stability of the peptide.

In an embodiment, the furan-peptides as taught herein may contain atleast two amino acids. Preferably, the furan-peptides as taught hereinmay contain from 3 to 5000 amino acids. For example, the furan-peptidesas taught herein may contain from 3 to 30 amino acids, for example, thefuran-peptides as taught herein may contain from 3 to 10, or from 10 to20, or from 20 to 30 amino acids. In certain embodiments, thefuran-peptides as taught herein may contain from 20 to 50 amino acids,or from 50 to 100 amino acids, or from 100 to 1000, or from 1000 to 5000amino acids. For example, the furan-peptides as taught herein maycontain at least 20, at least 25, at least 30, at least 35, at least 40,at least 45, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 200, at least 300, at least 400, atleast 500, at least 600, at least 700, at least 800, at least 900 or atleast 1000 amino acids.

In certain embodiments, the furan-peptides as taught herein may beunbound furan-peptides or furan-peptides bound to a solid support. Inpreferred embodiments, the furan-peptide as taught herein may be anunbound furan-peptide.

The terms “free”, “deprotected”, or “unbound” denote that the peptide isnot coupled to a solid support (e.g. the furan-peptide is cleaved fromthe solid support) on which it is synthesized or the furan-peptide isproduced by protein translation. The free or unbound furan-peptides astaught herein include, but are not limited to, furan-peptides insolution and dried or lyophilized furan-peptides, such as, for instancea powder of furan-peptides.

The furan-peptides as taught herein may be provided in dried orlyophilized form, such as, for instance a powder of furan-peptides.

The furan-peptides as taught herein may be provided in solution. Thefuran-peptides as taught herein may be provided in a solvent wherein thefuran-peptides can be dissolved. In certain embodiments, the solvent isa polar protic solvent (such as water, methanol, or ethanol), a polaraprotic solvent (such as DMSO, dimethylformamide (DMF), acetonitrile, ortetrahydrofuran (THF)), or a non-polar solvent (such as chloroform ordichloromethane (DCM)). In certain embodiments, the furan-peptides astaught herein are provided in a solvent comprising or consisting of apolar protic solvent (such as water, methanol, or ethanol), a polaraprotic solvent (such as DMSO, DMF, acetonitrile, or THF), or an apolarsolvent (such as chloroform or DCM).

The solid support for peptide synthesis is any support on which thefuran-peptides as taught herein are synthesized. The solid supports forpeptide synthesis may be polystyrene resins comprising an acid labilelinker, a base-labile linker, or a photo-labile linker, or any otherlinker known in the art. The solid supports for peptide synthesis mayalso be polyethylene glycol (PEG) resins comprising an acid labilelinker, a base-labile linker, or a photo-labile linker, or any otherlinker known in the art. The solid supports for peptide synthesis mayalso be polystyrene-co-polyethylene glycol resins comprising an acidlabile linker, a base-labile linker, or a photo-labile linker, or anyother linker known in the art. The solid supports for peptide synthesisare preferably chosen from polystyrene resins comprising an acid or baselabile linker or polystyrene-co-polyethylene glycol resins comprising anacid or base labile linker. In an embodiment, the solid support forpeptide synthesis may be selected from the group comprising Wang resin,Rink amide resin, ChemMatrix®, phenylacetamidomethyl (PAM) resin,Merrifield resin, and paramethyl-benzhydrylamine (pMBHA) resin. It willbe understood that furan-peptides which are subsequently, e.g. aftersynthesizing and cleaving from the solid support, coupled, connected orlinked to a further solid support, such as for instance beads,membranes, colloids, rubber or synthetic particles and the like, can beconsidered free furan-peptides.

The furan-peptides as taught herein may be provided in a composition. Inan embodiment, the composition preferably comprises or consists of atleast 60%, preferably, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% of furan-peptides. For example, the compositioncomprises from about 60% to about 70% of furan-peptides, for example,from about 70% to about 80% of furan-peptides, for example, from about80% to about 90% of furan-peptides, for example, the compositioncomprises from about 90% to about 100% of furan-peptides. In anembodiment, the composition consists of 100% of substantially purefuran-peptides.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be a GPCR and the ligand may be apeptide.

Accordingly, in certain embodiments, the present invention relates to amethod for covalently binding a GPCR (e.g., GPR54) and a peptide (e.g.,kisspeptin-10), the peptide being capable of specifically binding to theGPCR, the method consisting essentially of contacting living cellsexpressing the GPCR with the peptide comprising at least one furanmoiety, thereby covalently binding the GPCR and the peptide.

As mentioned above, in certain embodiments of the methods or cell-basedassays as taught herein, the ligand may be a peptide, a nucleoside, anucleic acid, a lipid, a polysaccharide, a small molecule, or acombination thereof. In certain embodiments, the ligand may be apeptide, a nucleoside, a nucleic acid, a lipid, or a small molecule. Incertain embodiments, the ligand may be a peptide, a nucleoside, anucleic acid, or a lipid. In certain embodiments, the ligand may be apeptide, a nucleoside, a nucleic acid, or a small molecule. In certainembodiments, the ligand may be a peptide, a nucleoside, or a nucleicacid.

In certain embodiments, the ligand as taught herein may be a nucleoside.Suitable non-limiting examples of nucleosides include cytidine,deoxycytidine, uridine, deoxyuridine, adenosine, deoxyadenosine,guanosine, deoxyguanosine, thymidine, 5-methyluridine, and inosine.

In certain embodiments, the ligand as taught herein may be a nucleicacid such as RNA or DNA.

In certain embodiments, a furan moiety may be incorporated in anucleoside (and thus in a nucleic acid) by methods known in the art. Forinstance, a furan moiety may be incorporated in a nucleoside byalkylation of the 2′-OH (Jawalekar et al., 2011, Chem. Commun., 47,2796-2798). Alternatively, a furan carboxylic acid can be coupledthrough amide bond formation to a 2′ amino nucleoside (Op de Beeck andMadder, 2012, JACS, 134, 10737-10740), or via reaction of a 2′-aminonucleoside with furfurylisocyanate (i.e., compound of Formula IIIi) (Opde Beeck and Madder, 2012, supra). Finally, a furan moiety can beintroduced in the 5-position of a nucleoside base through Stille crosscoupling of stannylated furan with commercially available5-iodo-2′-deoxyuridine (Carrette at al., 2013, Bioconjugate Chem.,2008-2014).

In certain embodiments, the ligand as taught herein may be a lipid.Suitable non-limiting examples of lipids include lysophosphatidic acidand sphingosine-1-phosphate.

In certain embodiments of the methods or cell-based assays, as taughtherein, the ligand may be lysophosphatidic acid and the cell surfaceprotein may be one or more, such as two, three, four, five or six,lysophospholipid receptors selected from lysophosphatidic acid receptor1 (LPAR1 or EDG2), lysophosphatidic acid receptor 2 (LPAR2 or EDG4),lysophosphatidic acid receptor 3 (LPAR3 or EDG7), lysophosphatidic acidreceptor 4 (LPAR4 or GPR23), lysophosphatidic acid receptor 5 (LPAR5 orGPR92), or lysophosphatidic acid receptor 6 (LPAR6 or P2RY5).

In certain embodiments of the methods or cell-based assays, as taughtherein, the ligand may be sphingosine-1-phosphate and the cell surfaceprotein may be one or more, such as two, three, four of five,lysophospholipid receptors selected from sphingosine-1-phosphatereceptor 1 (S1PR1 or EDG1), sphingosine-1-phosphate receptor 2 (S1PR2 orEDG5), sphingosine-1-phosphate receptor 3 (S1PR3 or EDG3),sphingosine-1-phosphate receptor 4 (S1PR4 or EDGE), orsphingosine-1-phosphate receptor 5 (S1PR5 or EDGE).

In certain embodiments, a furan moiety may be incorporated in a lipid bymethods known in the art. For instance, a furan moiety may beincorporated in a lipid via esterification of the lipid with a furanfatty acid. Furan fatty acids may occur as minor compounds in the lipidsof different food samples (Vetter and Wendlinger, 2013, LipidTechnology, 25, 7-10). Methods for the isolation of furan lipids andfuran fatty acids of foods, such as avocado, have been described, forinstance in U.S. Pat. No. 6,582,688.

In certain embodiments, the ligand as taught herein may be apolysaccharide.

In certain embodiments, a furan moiety may be incorporated in apolysaccharide by methods known in the art. For instance, a furan moietymay be incorporated in a polysaccharide via reaction of furan carboxylicacids or furfurylisocyanate with amino groups of aminoglycosidemoieties.

In certain embodiments, the ligand as taught herein may be a smallmolecule. In certain embodiments, the small molecule may be a drug, apesticide, or a cell signaling molecule.

In certain embodiments, a furan moiety may be incorporated in a smallmolecule by methods known in the art. For instance, a furan moiety maybe incorporated in a small molecule by reacting a small molecule with acommercially available furan derivative (e.g., compounds of Formula Ma,IIIb, IIIc, IIId, IIIe, IIIf, IIIh, IIIg, IIIi, IIIj, or IIIk as definedherein) For instance, depending on the functional group availability, acommercially available furan derivative such as furan carboxylic acidsor furfurylisocyanate can be incorporated into a small molecule bymethods known in the art such as esterification, amide bond formation,aryl-aryl coupling, or urea bond formation.

As mentioned above, a first aspect provides a method for covalentlybinding a cell surface protein and a ligand, the ligand being capable ofspecifically binding to the cell surface protein, the method consistingessentially of contacting living cells expressing the cell surfaceprotein with the ligand comprising at least one furan moiety, therebycovalently binding the cell surface protein and the ligand.

The cells as used in the methods or cell-based assays as taught hereinare living (viable) cells.

The terms “living cells” or “viable cells” as used herein refer to cellsthat can be qualified as viable by tests known per se, and moreparticularly refer to cells that are capable of dividing andproliferating. Where cells are said to be living or viable, a sizeablefraction of the tested cells may test as viable, e.g., at least about20%, at least about 40%, preferably at least about 50%, more preferablyat least about 60%, even more preferably at least about 70%, still morepreferably at least about 80%, yet more preferably at least about 90%,and still more preferably at least about 95% and up to 100% of thetested cells may test as viable.

Viability of cells may be measured using techniques known in the art.Techniques for determining viability or cell survival are commonlyreferred to as viability assays. For instance, the viability of cellsmay be measured using conventional dye exclusion assays, such as TrypanBlue exclusion assay or propidium iodide exclusion assay. In suchassays, viable cells exclude the dye and hence remain unstained, whilenon-viable cells take up the dye and are stained. The cells and theiruptake of the dye can be visualised and revealed by a suitable technique(e.g., conventional light microscopy, fluorescence microscopy, flowcytometry), and viable (unstained) and non-viable (stained) cells in thetested sample can be counted. Cell survival can be convenientlyexpressed as the absolute number of living cells, or as cell viability(i.e., the ratio or proportion (%) of viable cells to total (i.e., sumof viable and non-viable) cells).

In certain embodiments, the cells as taught herein may be animal cells,preferably warm-blooded animal cells, more preferably mammalian cells,such as human cells or non-human mammalian cells, and most preferablyhuman cells.

In certain embodiments, the cells as intended herein may be cellspresent in vivo, such as cells in tissues or bodily fluids presentinside the body.

In certain embodiments, the cells as intended herein may be cellspresent ex vivo, such as cells in tissues or homogenized tissues.

In certain embodiments, the cells as intended herein may be cells grownor cultured in vitro. Preferably, the cells may be cells grown orcultured in vitro.

In certain embodiments, the cells as taught herein may be obtained froma biological sample of a subject.

The term “subject” or “patient” are used interchangeably and refer toanimals, preferably warm-blooded animals, more preferably vertebrates,and even more preferably mammals specifically including humans andnon-human mammals, that have been the object of treatment, observationor experiment. The term “mammal” includes any animal classified as such,including, but not limited to, humans, domestic and farm animals, zooanimals, sport animals, pet animals, companion animals and experimentalanimals, such as, for example, mice, rats, hamsters, rabbits, dogs,cats, guinea pigs, cattle, cows, sheep, horses, pigs and primates, e.g.,monkeys and apes. Particularly preferred are human subjects, includingboth genders and all age categories thereof.

Non-human animal subjects may also include prenatal forms of animals,such as, e.g., embryos or foetuses. Human subjects may also includefoetuses, but by preference not embryos.

The term “biological sample” or “sample” as used herein generally refersto a sample obtained from a biological source, e.g., from an organism,organ, tissue or cell culture, etc. A biological sample of an animal orhuman subject refers to a sample removed from an animal or human subjectand comprising cells thereof. The biological sample of an animal orhuman subject may comprise one or more tissue types and cells of one ormore tissue types. Methods of obtaining biological samples of an animalor human subject are well known in the art, e.g., tissue biopsy ordrawing blood.

In an embodiment, the cells may be obtained from a healthy subject. Thismay advantageously allow identifying and/or studying cell surfaceprotein-ligand interactions in a healthy subject.

In another embodiment, the cells may be obtained from a diseasedsubject. In certain embodiments, the cells may be obtained from asubject who has a proliferative disease or disorder, for instance atumour or cancer. In certain embodiments, the cells may be obtained froma subject who has an infectious disease. In certain embodiments, thecells may be obtained from a subject who has a genetic disease. Incertain embodiments, the cells may be obtained from a subject who has ametabolic disease. In certain embodiments, the cells may be obtainedfrom a subject who has a respiratory disease. In certain embodiments,the cells may be obtained from a subject who has a rare disease. Incertain embodiments, the cells may be obtained from a subject who has anauto-immune disease.

The term “proliferative disease or disorder” generally refers to anydisease or disorder characterized by neoplastic cell growth andproliferation, whether benign, pre-malignant, or malignant. The termproliferative disease generally includes all transformed cells andtissues and all cancerous cells and tissues. Proliferative diseases ordisorders include, but are not limited to abnormal cell growth, benigntumours, premalignant or precancerous lesions, malignant tumours, andcancer.

As used herein, the terms “tumour” or “tumour tissue” refer to anabnormal mass of tissue resulting from excessive cell division. A tumouror tumour tissue comprises “tumour cells” which are neoplastic cellswith abnormal growth properties and no useful bodily function. Tumours,tumour tissue and tumour cells may be benign, pre-malignant ormalignant, or may represent a lesion without any cancerous potential. Atumour or tumour tissue may also comprise “tumour-associated non-tumourcells”, e.g., vascular cells which form blood vessels to supply thetumour or tumour tissue, or immune cells as part of the tumourmicroenvironment. Non-tumour cells may be induced to replicate anddevelop by tumour cells, for example, the induction of angiogenesis in atumour or tumour tissue.

As used herein, the term “cancer” refers to a malignant neoplasmcharacterized by deregulated or unregulated cell growth.

The term “infectious disease” generally refers to any disease resultingfrom an infection.

The term “infection” generally refers to an invasion of a subject's bodytissues by disease-causing agents, their multiplication, and thereaction of the subject's tissues to these organisms and the toxins theyproduce. Disease-causing agents or infectious agents include bacteria;viruses; viroids; prions; nematodes such as parasitic roundworms andpinworms; arthropods such as ticks, mites, fleas, and lice; fungi suchas ringworm; and other macroparasites such as tapeworms and otherhelminths.

In certain embodiments, the cells as taught herein may be primary cells.In certain embodiments, the cells as taught herein may be obtained froma primary cell culture.

The term “primary cells” generally refers to cells that are grown orcultured directly after isolation from a subject.

The term “primary cell culture” refers to a cell culture obtained bygrowing or culturing cells directly after their isolation from asubject. Primary cell cultures typically have a limited lifespan.

In certain embodiments, the cells as taught herein may be obtained froman established cell line.

The terms “established cell line” or “immortalized cell line” generallyrefer to a cell line that has acquired the ability to proliferateindefinitely (for instance through random mutation or deliberatemodification, such as artificial expression of the telomerase gene).

Established cell lines and primary cells are commercially available, forinstance from the ATCC Cell Biology Collection, the European Collectionof Authenticated Cell Cultures (ECACC), or the Biobank (CreativeBioarray, NY, USA).

In certain embodiments, the cells as taught herein may be obtained froma human cell line. In certain embodiments, the cells as taught hereinmay be obtained from a non-human mammalian cell line, such as a mousecell line.

In certain embodiments, the cells as taught herein may be obtained froma cell line derived from normal (healthy) tissue or cells. In certainembodiments, the cells as taught herein may be obtained from a cell linederived from a diseased tissue or cells.

In certain embodiments, the cells as taught herein may be obtained froma normal (healthy) cell line. In certain embodiments, the cells astaught herein may be obtained from a cancer cell line, a tumour cellline, an infectious disease cell line, a metabolic disease cell line, agenetic disease cell line, a respiratory disease cell line, or a raredisease cell line.

Suitable non-limiting examples of normal human cell lines include humanumbilical vein endothelial cells (HUVECs), T- or B-cells form healthydonors, human aortic smooth muscle cells (HAOSMCs), human bronchialepithelial cells (HBEpCs), human mammary epithelial cells (HMEpC), andhuman lung cell lines such as MRC-5.

Suitable non-limiting examples of normal mouse cell lines include NIH3T3 fibroblasts, mouse embryonic fibroblasts (MEFs), Ba/F3 B-cell line,mouse T-cells, and Sol 8 myoblast cell line.

Suitable non-limiting examples of cancer human cell lines includeMDA-MB-231 (breast cancer), MCF-7 (breast cancer), HeLa (cervix cancer),PC3 (prostate cancer), HEPG2 (liver cancer), Jurkat (leukemia), andHT1080 (fibrosarcoma).

Suitable non-limiting examples of cancer mouse cell lines includeHEPA1-6 (liver cancer), 4T1 (breast cancer), Tramp C1-3 (prostatecancer), and B16-F10 (melanoma).

In certain embodiments of the methods or cell-based assays as taughtherein, the living cells as taught herein may be normal cells (healthycells). In certain embodiments, the cells as taught herein may bediseased cells such as cancerous cells (e.g., derived from a cancer cellline or cancer or tumour) or infected cells (e.g., derived from aninfectious disease cell line or infected tissue).

In certain preferred embodiments, the cells as taught herein may benormal cells. The present inventors surprisingly found that the methodsof the present invention allow covalent binding of a cell surfacereceptor and a ligand specifically binding to the cell surface receptorin normal cells using only the cell's own biosynthetic machinery. Thisis completely unexpected since in normal cells (as opposed to diseasedcells) the covalent binding of the cell surface receptor and the ligandcannot be attributed to the presence of oxidative stress. The precisenature of the cellular source causing the endogenous activation remainsunknown.

When the methods are performed in vitro, the living cells may be grownas an adherent monolayer on a surface or the living cells may be in cellsuspension.

In certain embodiments, the cells as taught herein may be adherent,i.e., require a surface for growth, and typically grow as an adherentmonolayer on said surface (i.e., adherent cell culture). Adhesion ofcells to a surface, such as the surface of a tissue culture plasticvessel, can be readily examined by visual inspection under invertedmicroscope. Cells grown in adherent culture require periodic passaging,wherein the cells may be removed from the surface enzymatically (e.g.,using trypsin), suspended in growth medium, and re-plated into newculture vessel(s). In general, a surface or substrate which allowsadherence of cells thereto may be any substantially hydrophilicsubstrate. As known in the art, tissue culture vessels, e.g., cultureflasks, well plates, dishes, or the like, may be usually made of a largevariety of polymeric materials, suitably surface treated or coated aftermoulding in order to provide for hydrophilic substrate surfaces.

In certain embodiments, the cells as taught herein may be free-floatingcells in a culture medium (suspension culture). The terms “suspension”and “cell suspension” generally refers to a heterogeneous mixturecontaining cells dispersed in a liquid phase. As the composition isliquid, the cells may in principle be able to, but need not, settle orsediment from the composition.

In an embodiment, the methods as taught herein consist essentially of orconsist of contacting (or bringing together) living cells expressing acell surface protein with a ligand comprising at least one furan moiety.

The term “contacting living cells expressing a cell surface protein witha ligand” as used herein refers to exposing the living cells expressingthe cell surface protein to the ligand. The living cells expressing thecell surface protein are contacted with the ligand to ensure that theligand can specifically bind to the cell surface protein expressed onthe living cells.

In certain embodiments, the living cells and the ligand may be contactedfor at least 1 minute. For instance, the living cells and the ligand maybe contacted for at least 2 minutes, at least 3 minutes, at least 4minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes,at least 20 minutes, at least 25 minutes, at least 30 minutes, at least35 minutes, at least 40 minutes, at least 45 minutes, at least 50minutes, at least 55 minutes, at least 60 minutes (1 hour), at least 90minutes (1.5 hours), at least 120 minutes (2 hours), at least 150minutes, at least 180 minutes (3 hours), at least 240 minutes (4 hours),at least 300 minutes (5 hours), or at least 360 minutes (6 hours).Preferably, the living cells and the ligand may be contacted for atleast 5 minutes such as for 10 minutes, 15 minutes, 30 minutes or 60minutes (1 hour).

Time, as used herein, is expressed in minutes or hours. It is to benoted however that time may also be expressed in other units such asseconds.

The terms “time”, “time period”, and “time duration” may be usedinterchangeably.

In certain embodiments, the living cells and the ligand may be contactedunder physiological conditions. In certain embodiments of the methods orcell-based assays as taught herein, the methods or cell-based assays astaught herein may be performed under physiological conditions.Advantageously, such conditions allow the application of the methods orcell-based assays as described herein in a cellular context, e.g., onadherent living cells or on living cells in suspension.

In certain embodiments, the methods or cell-based assays as taughtherein may be performed in solution. The methods or cell-based assays astaught herein may be performed in a solution which is compatible withcell viability and in which the ligand is soluble. The solution may beany physiological solution such as cell culture media covering adherentcells or cell culture media in which cells are in suspension.

In certain embodiments, the methods or cell-based assays as describedherein may be performed in an aqueous solution. In certain preferredembodiments, the living cells and the ligand may be contacted in anaqueous solution. As mentioned above, in certain embodiments, theligands as taught herein may be provided in a solvent. In certainembodiments of the methods or cell-based assays as taught herein, thefinal concentration of the solvent in the aqueous solution is kept belowa concentration that is toxic to the cells (e.g., for ligands dissolvedin DMSO, the concentration of DMSO is kept below 0.5%, preferably below0.2%, more preferably below 0.1%). Such final concentrations of thesolvent in the aqueous solution do not compromise the viability orproliferation of the living cells.

The term “aqueous solution” generally refers to a solution in which thesolvent comprises, consists essentially of, or consists of water. Incertain embodiments, the aqueous solution comprises at least 0.1% ofwater. For example, the aqueous solution comprises at least 0.5%, atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 99% of water. In certainembodiments, the solvent consists of water.

In certain embodiments, the aqueous solution may be a buffer such as aphosphate buffer, preferably phosphate buffered saline (PBS).

In certain embodiments, the aqueous solution may be cell culture mediaas known per se, such as for example liquid cell culture media.Well-known cell culture media include Dulbecco's Modified Eagles Medium(DMEM) such as high glucose (4.5 mg/ml) DMEM or low glucose (1 mg/ml)DMEM; Eagle's minimal essential medium (EMEM); Roswell Park MemorialInstitute (RPMI) media; and Ham's F12 media. The cell culture media mayoptionally comprise GlutaMax™. The cell culture media may comprise serumsuch as Fetal bovine serum (FBS) or Fetal calf serum (FCS). The cellculture media may comprise serum in a concentration of at most 10%, suchas at a concentration of 0.5%, 1.0%, 5.0% or 10%.

In certain preferred embodiments, the methods or cell-based assays asdescribed herein may be performed in cell culture media, such as DMEM,EMEM, and RPMI media, optionally comprising serum, such as FBS or FCS.This advantageously allows performing the methods or cell-based assaysas taught herein in the cell culture media in which the cells have beengrown. In certain embodiments, the living cells and the ligand may becontacted in an aqueous solution selected from a buffer such as aphosphate buffer, preferably PBS, or culture media such as DMEM, EMEM,and RPMI media.

In certain embodiments of the methods or cell-based assays as taughtherein, the ligand as taught herein may be provided in an aqueoussolution. In certain embodiments, the living cells expressing the cellsurface protein as taught herein may be provided in an aqeuous solution.

In certain embodiments, the methods or cell-based assays as describedherein may be performed in an aqueous solution without the use oforganic solvents. In certain embodiments, the methods or cell-basedassays as described herein may be performed in an aqueous solutionwithout the use of reducing agents. In certain embodiments, the methodsor cell-based assays as described herein may be performed without theuse of toxic additives (e.g., copper or aniline) and/or without the useof catalysts (e.g., copper or aniline) In certain embodiments, themethods or cell-based assays as described herein may be performed in anaqueous solution without the use of organic solvents and/or without theuse of reducing agents and/or without the use of toxic additives and/orwithout the use of catalysts. Such conditions advantageously offer greatpotential for the applications of the methods or cell-based assays asdescribed herein.

In certain embodiments, the methods or cell-based assays as describedherein may be performed at a pH ranging from about 6 to about 8. Incertain embodiments, the methods or cell-based assays as describedherein may be performed at a near neutral pH (i.e., pH of about 7).

In certain embodiments, the methods or cell-based assays as describedherein may be performed at a temperature ranging from about 0° C. toabout 40° C., or from about 4° C. to about 40° C., or from about 10° C.to about 40° C., or from about 20° C. to about 40° C. In certainembodiments, the methods or cell-based assays as described herein may beperformed at a temperature of about 37° C.

Temperature, as used herein, is expressed in degrees Celsius (° C.). Itis to be noted however that temperature may also be expressed in anyother suitable unit such as Kelvin (K).

In certain embodiments, the methods or cell-based assays as describedherein may be performed in an aqueous solution, at near neutral pH, andat a temperature of about 37° C. Such conditions allow the applicationof the methods or cell-based assays as described herein under conditionswhich do not influence cell viability or cell functioning both in vitroand in vivo. This is in contrast to prior art methods such asphotoaffinity crosslinking which require working with cell lysates or,when working with living cells, in cold buffers.

In certain embodiments, the concentration of the ligand in the aqueoussolution may be at least 0.01 μM. In certain embodiments, theconcentration of the ligand in the aqueous solution may be at least 0.1μM, at least 0.5 μM, at least 1.0 μM, at least 5.0 μM, at least 10.0 μM,or at least 50.0 μM.

In certain embodiments, the concentration of the ligand in the aqueoussolution may be at ranging from 0.1 μM to 100.0 μM. In certainembodiments, the concentration of the ligand in the aqueous solution maybe ranging from 0.5 μM to 50.0 μM, or from 1.0 μM to 10.0 μM, or from0.1 μm to 10.0 μM, or from 0.1 μM to 5.0 μM.

In certain embodiments, the living cells may be grown as an adherentmonolayer on a surface. In certain embodiments, the living cells may begrown as an adherent monolayer on a surface to at least 50% confluency.In certain embodiments, the living cells may be grown as an adherentmonolayer on a surface to at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, such as 100% confluency.

In certain embodiments of the methods or cell-based assays as taughtherein, the number of the living cells contacted with the ligand may bebetween about 1×10³ and about 1×10¹¹, or between about 1×10⁴ and about1×10¹⁰, or between about 1×10⁵ and about 1×10⁹ cells. In certainembodiments, the number of the living cells contacted with the ligandmay be between about 1×10⁵ and about 1×10⁷ cells. In certainembodiments, the number of the living cells contacted with the ligandmay be between about 1×10⁵ and about 5×10⁶ cells.

In an embodiment, the method as taught herein may consist essentially ofor may consist of contacting living cells expressing the cell surfaceprotein with the ligand comprising at least one furan moiety in anaqueous solution, wherein the living cells are grown as an adherentmonolayer on a surface and the concentration of the ligand in theaqueous solution is ranging from 0.01 μM to 100.0 μM. For instance, themethod as taught herein may consist essentially of or may consist ofcontacting living cells expressing the cell surface protein with theligand comprising at least one furan moiety in an aqueous solution,wherein the living cells are grown as an adherent monolayer on asurface, and the concentration of the ligand in the aqueous solution isranging from 0.1 μM to 100.0 μM, from 0.5 μM to 50.0 μM, from 1.0 μM to10.0 μM, from 0.1 μm to 10.0 μM, or from 0.1 μM to 5.0 μM. Preferably,the method as taught herein may consist essentially of or may consist ofcontacting living cells expressing the cell surface protein with theligand comprising at least one furan moiety in an aqueous solution,wherein the living cells are grown as an adherent monolayer on asurface, and the concentration of the ligand in the aqueous solution isranging from 0.1 μM to 5.0 μM.

In certain embodiments of the methods or cell-based assays as taughtherein, the furan moiety (of the ligand) may be oxidized by endogenousactivation. The endogenous activation advantageously allows thecovalently binding of a cell surface protein and a ligand without theneed for any exogenous (chemical or physical) activation.

As used herein the term “endogenous” refers to originating from orproduced by a cell, tissue, or organism.

As used herein the term “exogenous” refers to caused or produced byfactors external to a cell, tissue, or organism.

In certain embodiments, the furan moiety (of the ligand) is oxidized bythe cells. In certain embodiments, the furan moiety (of the ligand) isoxidized by the cells through endogenous activation. In certainembodiments, the furan moiety (of the ligand) is oxidized by a cellularsource.

In certain embodiments, the furan moiety (of the ligand) is oxidized bya cellular source through endogenous activation. The precise nature ofthe cellular source is unknown.

The present inventors have found that when living cells expressing acell surface protein are contacted with a ligand which specificallybinds the cell surface protein, covalent binding between the cellsurface protein and the ligand occurs. This suggests that specificbinding of the ligand to the cell surface protein on the living cells issufficient to cause activation of the furan moiety.

In certain embodiments of the methods or cell-based assays as taughtherein, the endogenous activation may occur at the extracellular spaceof the cell membrane.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may comprise at least one amine group,hydroxyl group, sulfhydryl group, imidazole group and/or indole group.In certain embodiments of the methods or cell-based assays as taughtherein, the oxidized furan moiety of the ligand may react with the aminegroup, hydroxyl group, sulfhydryl group, imidazole group and/or indolegroup of the cell surface protein. In certain embodiments, the furanmoiety of the ligand may be oxidized by endogenous activation, the cellsurface protein may comprise at least one amine group, hydroxyl group,sulfhydryl group, imidazole group and/or indole group, and the oxidizedfuran moiety of the ligand may react with the amine group, hydroxylgroup, sulfhydryl group, imidazole group and/or indole group of the cellsurface protein.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may comprise a binding site comprisingat least one nucleophile. Such a nucleophile advantageously allowsbinding with the oxidized furan moiety. Hence, in certain embodiments,the oxidized furan moiety of the ligand may react with the nucleophileof the binding site of the cell surface protein. In certain embodiments,the furan moiety of the ligand may be oxidized by endogenous activation,the cell surface protein may comprise a binding site comprising anucleophile, and the oxidized furan moiety of the ligand may react withthe nucleophile of the binding site of the cell surface protein.

In certain embodiments of the methods or cell-based assays as taughtherein, the nucleophile may be an amine group, a hydroxyl group, asulfhydryl group, an imidazole group and/or an indole group. In certainembodiments, the cell surface protein may comprise a binding sitecomprising at least one amine group, hydroxyl group, sulfhydryl group,imidazole group and/or indole group. Cell surface proteins comprising abinding site comprising a nucleophile, such as amine group, hydroxylgroup, sulfhydryl group, imidazole group and/or indole group,advantageously allow the crosslinking of the cell surface protein andthe ligand.

In certain embodiments, the furan moiety (of the ligand) is oxidizedwithout the addition of an exogenous activation signal. In certainembodiments, the furan moiety (of the ligand) is oxidized without theaddition of an exogenous oxidative reagent. The example sectionillustrates that the present methods allow for the first time chemicalcrosslinking (covalent binding) of a (modified, i.e., furan-containing)ligand on its unmodified endogenous receptor in cells without any formof exogenous intervention (e.g., chemical activation signal such as NBS, or physical activation signal such as UV-light).

The methods illustrating the present invention allow covalent in situlabeling of cell surface proteins, e.g. in diagnostics, as a cheap,reliable alternative to antibodies. The present methods allow thedetection and/or in situ visualization of cell surface proteins, therebyallowing to study internalization, trafficking and/or diffusioncharacteristics of the cell surface proteins in the cell membrane.

A further aspect relates to a method for detecting a cell surfaceprotein covalently bound to a ligand, the ligand being capable ofspecifically binding to the cell surface protein, the method consistingessentially of or consisting of:

-   -   performing the methods as taught herein, and    -   detecting the cell surface protein covalently bound to the        ligand.

Accordingly, certain embodiments provide a method or cell-based assayfor detecting a cell surface protein covalently bound to a ligand, theligand being capable of specifically binding to the cell surfaceprotein, the method consisting essentially of or consisting ofcontacting living cells expressing the cell surface protein with theligand comprising at least one furan moiety, thereby covalently bindingthe cell surface protein and the ligand, and detecting the cell surfaceprotein covalently bound to the ligand.

By performing the methods as taught herein, the cell surface protein andthe ligand are covalently bound. Optionally, the ligand may comprise alabel, which may facilitate the detection (e.g., visualization) of thecell surface protein. The step of detecting the cell surface proteincovalently bound to the ligand may be performed by any adequatetechnique known to the skilled person. In certain embodiments, the stepof detecting the cell surface protein covalently bound to the ligand maybe performed by flow cytometry such as Fluorescence-activated cellsorting (FACS); by microscopy such as fluorescence confocal microscopy,epifluorescence microscopy, or live cell imaging; gel-electrophoresis;Western blot; immunoassays such as enzyme-linked immunosorbent assay(ELISA); mass spectrometry such as matrix-assisted laserdesorption/ionization-time of flight (MALDI-TOF), electro sprayionization-mass spectrometry (ESI-MS), liquid chromatography-massspectrometry (LC-MS), and orbitrap mass spectrometry; or a combinationthereof.

The wordings “detecting the cell surface protein covalently bound to theligand”, “determining the presence of a covalently bound complex of thecell surface protein and the ligand” and “determining the covalent bondbetween the ligand and the cell surface protein” may be usedinterchangeably herein.

The methods of the invention may be used to identify, for a knownligand, a cell surface protein to which the ligand is capable ofspecifically binding thereto. Hence, in an embodiment, the presentinvention relates to the use of the methods for covalently binding acell surface protein and a peptide as taught herein, for identifying,for a known ligand, a cell surface protein to which the ligand iscapable of specifically binding thereto. The present inventors furtherfound that the cell-based assays as taught herein allow identifyingtarget cell surface proteins of biologically active orphan ligands, suchas orphan peptides or small molecules, without the requirement forexogenous activation. For instance, the present cell-based assays allowto screen peptide libraries generated by standard solid phase peptidesynthesis and to identify the cell surface receptors via massspectrometry-based sequencing if a covalently bound complex is present.

Accordingly, a further aspect provides a cell-based assay foridentifying, for a known ligand, a cell surface protein to which theligand is capable of specifically binding thereto, the ligand comprisingat least one furan moiety, the cell-based assay consisting essentiallyof or consisting of:

-   -   contacting living cells with the ligand;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand; and    -   identifying the cell surface protein if the covalently bound        complex is present.

In certain embodiments, the step of determining the presence of acovalently bound complex of the cell surface protein and the ligand maybe performed by the techniques as described herein.

The step of identifying the cell surface protein may be performed by anyadequate technique known to the skilled person for protein massspectrometry (MS) analysis, preferably by gel electrophoresis, gaschromatography-mass spectrometry (GC-MS), liquid chromatography-massspectrometry (LC-MS), high performance liquid chromatography-massspectrometry (HPLC-MS), reversed phase high performance liquidchromatography-mass spectrometry (RP HPLC-MS), matrix-assisted laserdesorption/ionization-time of flight (MALDI-TOF), electro sprayionization-mass spectrometry (ESI-MS), inductively coupled plasma-massspectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermalionization-mass spectrometry (TI-MS), orbitrap mass spectrometry, orspark source mass spectrometry (SS-MS), more preferably by gelelectrophoresis, liquid chromatography-mass spectrometry (LC-MS),matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF),or electro-spray ionization-mass spectrometry (ESI-MS). These techniquesfor protein mass spectrometry analysis can optionally be preceded by anenzyme proteolytic digest such as a tryptic digest.

RP-HPLC is generally used to monitor the reaction progress according tothe retention time of the analytes which is related to the polarity ofthe analytes.

MALDI-TOF analysis may provide information on the mass of the moleculesinvolved.

Preferably, the step of identifying the cell surface protein isperformed using HPLC-MS.

Furthermore, the methods as taught herein may be used to identify, for aknown cell surface protein, a ligand specifically binding to the cellsurface protein. Hence, in an embodiment, the present invention relatesto the use of the methods for covalently binding a cell surface proteinand a ligand as taught herein, for identifying, for a known cell surfaceprotein, a ligand specifically binding to the cell surface protein. Thepresent inventors realized that the cell-based assays as taught hereinallow identifying for a known cell surface protein a ligand whichspecifically binds the cell surface protein by screening ligandlibraries and inferring from the finding that a covalently bound complexof the cell surface protein and the ligand is present, that the ligandspecifically binds the cell surface protein.

Hence, a further aspect relates to a cell-based assay for identifying,for a known cell surface protein, a ligand specifically binding to thecell surface protein, the cell-based assay consisting essentially of orconsisting of:

-   -   contacting living cells expressing the cell surface protein with        a ligand comprising at least one furan moiety;    -   determining the presence of a covalently bound complex of the        cell surface protein and the ligand; and    -   inferring from the finding that the covalently bound complex is        present that the ligand specifically binds the cell surface        protein.

The methods of the invention may be used to identify a binding site ofan interacting peptide-cell surface protein pair. Hence, in anembodiment, the present invention relates to the use of the methods forcovalently binding a cell surface protein and a peptide as taughtherein, for identifying a binding site of a cell surface protein and apeptide. The present cell-based assays can be used to gain immediateinsight on the location of the peptide-cell surface protein bindingsite.

Accordingly, a further aspect relates to a cell-based assay foridentifying a binding site of a cell surface protein and a peptide, thepeptide being capable of specifically binding to the cell surfaceprotein and the peptide comprising at least one amino acid comprising afuran moiety, wherein said amino acid comprising a furan moiety islocated at position n of the peptide, the cell-based assay consistingessentially of or consisting of:

-   (a) contacting living cells with the peptide;-   (b) determining the presence of a covalently bound complex of the    cell surface protein and the peptide;-   (c) identifying the amino acid comprising a furan moiety as a    binding site of the cell surface protein and the peptide if the    covalently bound complex is present;-   (d) optionally repeating steps (a) to (c) with peptides comprising a    furan moiety, wherein the amino acid comprising a furan moiety is    located at position n+p of the peptides comprising a furan moiety;    wherein position n may be any amino acid of the peptides comprising    a furan moiety, and wherein p is a positive or negative integer    (provided position n+p is located on the peptides comprising a furan    moiety).

In certain embodiments of the methods or cell-based assays as taughtherein, the ligand as taught herein may be a peptide.

Accordingly, in certain embodiments, the present invention relates to acell-based assay for identifying, for a known peptide, a cell surfaceprotein to which the peptide is capable of specifically binding thereto,the peptide comprising at least one furan moiety, the cell-based assayconsisting essentially of or consisting of:

-   -   contacting living cells with the peptide;    -   determining the presence of a covalently bound complex of the        cell surface protein and the peptide; and    -   identifying the cell surface protein if the covalently bound        complex is present.

In certain embodiments, the present invention relates to a cell-basedassay for identifying, for a known cell surface protein, a peptidespecifically binding to the cell surface protein, the cell-based assayconsisting essentially of or consisting of:

-   -   contacting living cells expressing the cell surface protein with        a peptide comprising at least one furan moiety;    -   determining the presence of a covalently bound complex of the        cell surface protein and the peptide; and    -   inferring from the finding that the covalently bound complex is        present that the peptide specifically binds the cell surface        protein.

In certain embodiments of the methods or cell-based assays as taughtherein, the cell surface protein may be a GPCR and the ligand may be apeptide.

Accordingly, in certain embodiments, the present invention relates to acell-based assay for identifying, for a known peptide, a GPCR to whichthe peptide is capable of specifically binding thereto, the peptidecomprising at least one furan moiety, the cell-based assay consistingessentially of or consisting of:

-   -   contacting living cells with the peptide;    -   determining the presence of a covalently bound complex of the        GPCR and the peptide; and    -   identifying the GPCR if the covalently bound complex is present.

In certain embodiments, the present invention relates to a cell-basedassay for identifying, for a known GPCR, a peptide specifically bindingto the GPCR, the cell-based assay consisting essentially of orconsisting of:

-   -   contacting living cells expressing the GPCR with a peptide        comprising at least one furan moiety;    -   determining the presence of a covalently bound complex of the        GPCR and the peptide; and    -   inferring from the finding that the covalently bound complex is        present that the peptide specifically binds the GPCR.

In certain embodiments, the present invention relates to a cell-basedassay for identifying a binding site of a GPCR and a peptide, thepeptide being capable of specifically binding to the GPCR and thepeptide comprising at least one amino acid comprising a furan moiety,wherein said amino acid comprising a furan moiety is located at positionn of the peptide, the cell-based assay consisting essentially of orconsisting of:

-   (a) contacting living cells with the peptide;-   (b) determining the presence of a covalently bound complex of the    GPCR and the peptide;-   (c) identifying the amino acid comprising a furan moiety as a    binding site of the GPCR and the peptide if the covalently bound    complex is present;-   (d) optionally repeating steps (a) to (c) with peptides comprising a    furan moiety, wherein the amino acid comprising a furan moiety is    located at position n+p of the peptides comprising a furan moiety;    wherein position n may be any amino acid of the peptides comprising    a furan moiety, and wherein p is a positive or negative integer    (provided position n+p is located on the peptides comprising a furan    moiety).

A further aspect relates to an expression cassette or an expressionvector comprising a nucleic acid molecule encoding a cell surfaceprotein as defined herein and a promoter operably linked to the nucleicacid molecule. Preferably, the expression cassette or expression vectormay be configured to effect expression of the cell surface protein in ananimal cell, such as in a mammalian cell, including human cells andnon-human mammalian cells. In certain embodiments, the expressioncassette or expression vector is configured to effect expression of thecell surface protein in a human cell.

The terms “expression vector” or “vector” as used herein refers tonucleic acid molecules, typically DNA, to which nucleic acid fragmentsmay be inserted and cloned, i.e., propagated. Hence, a vector willtypically contain one or more unique restriction sites, and may becapable of autonomous replication in a defined host cell or vehicleorganism such that the cloned sequence is reproducible. A vector mayalso preferably contain a selection marker, such as, e.g., an antibioticresistance gene, to allow selection of recipient cells that contain thevector. Vectors may include, without limitation, plasmids, phagemids,bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleicacids, e.g., linear DNA, viral vectors, etc., as appropriate (see, e.g.,Sambrook et al., 1989; Ausubel 1992). Expression vectors are generallyconfigured to allow for and/or effect the expression of nucleic acids orORFs introduced thereto in a desired expression system, e.g., in vitro,in a host cell, host organ and/or host organism. For example, expressionvectors may advantageously comprise suitable regulatory sequences.

A further aspect provides a kit comprising: a ligand as taught hereincomprising at least one furan moiety; an expression vector comprising anucleic acid molecule encoding a cell surface protein as taught herein;and components or instructions for covalently binding the cell surfaceprotein and the ligand according to the methods as taught herein. Inembodiments, the kits may comprise: a ligand as taught herein comprisingat least one furan moiety; an expression vector comprising a nucleicacid molecule encoding a cell surface protein as taught herein; andinstructions for covalently binding the cell surface protein and theligand according to the methods as taught herein. In particularembodiments, the kits may comprise instructions for covalently bindingthe cell surface protein and the ligand according to the methods astaught herein.

In certain embodiments, the kits may comprise: a peptide comprising atleast one furan moiety; an expression vector comprising a nucleic acidmolecule encoding a cell surface protein as taught herein; andcomponents or instructions for covalently binding the cell surfaceprotein and the peptide according to the methods as taught herein. Incertain embodiments, the kits may comprise: a peptide comprising atleast one furan moiety; an expression vector comprising a nucleic acidmolecule encoding a cell surface protein as taught herein; andinstructions for covalently binding the cell surface protein and thepeptide according to the methods as taught herein. In particularembodiments, the kits may comprise instructions for covalently bindingthe cell surface protein and the peptide according to the methods astaught herein.

A further aspect provides a kit comprising a ligand comprising at leastone furan moiety, living cells expressing a cell surface protein, andcomponents or instructions for covalently binding the cell surfaceprotein and the ligand according to the methods as taught herein. Inembodiments, the kits comprise a ligand comprising at least one furanmoiety, living cells expressing a cell surface protein, and instructionsfor covalently binding the cell surface protein and the ligand accordingto the methods as taught herein. In particular embodiments, the kitscomprise instructions for covalently binding the cell surface proteinand the ligand according to the methods as taught herein.

In certain embodiments, the kits comprise a peptide comprising at leastone furan moiety, living cells expressing a cell surface protein, andcomponents or instructions for covalently binding the cell surfaceprotein and the peptide according to the methods as taught herein. Incertain embodiments, the kits comprise a peptide comprising at least onefuran moiety, living cells expressing a cell surface protein, andinstructions for covalently binding the cell surface protein and thepeptide according to the methods as taught herein. In particularembodiments, the kits comprise instructions for covalently binding thecell surface protein and the peptide according to the methods as taughtherein.

EXAMPLES

Materials and Methods

Materials

Fmoc-ß-(2-furyl)-Ala-OH, Fmoc-L-4-Benzoylphenylalanine-OH were purchasedfrom Peptech. All other amino acids, as well as Rink Amide AM (200-400mesh), coupling reagent2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), trifluoroacetic acid (TFA) and biotin-dPEG(4)-COOH werepurchased from Iris Biotech. ChemMatrix was obtained from Biotage.D-biotin was purchased from Chem-Impex International. Coupling reagent1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeniumhexafluorophosphate (COMU) was purchased from Novabiochem. Peptidesynthesis grade DMF was purchased from Biosolve. Dichloromethane,methanol, N,N-Diisopropylethylamine (DIPEA), dimethylsulfoxide (DMSO)and triisopropylsilane (TIS) were obtained from Sigma Aldrich.

Rabbit anti-GPR54 was purchased from Alomone Labs (cat. nr. AKR-001).IRDye conjugated streptavidin and secondary antibodies were obtainedfrom Li-Cor Biotechnology. Dulbecco's Modified Eagle Medium (DMEM),Dulbecco's Phosphate Buffered Saline (DPBS) and Fetal bovine serum (FBS)were purchased from Gibco.

N-bromosuccinimide, N-acetyl cysteine, and streptavidin-agarose resinwere obtained from Sigma Aldrich.

Peptide Synthesis

The different kisspeptin-10 peptides were synthesized by standardFmoc-based solid-phase peptide synthesis on a Syro II automated peptidesynthesizer (Multisyntech), using either Rink Amide AM (200-400 mesh) orRink Amide ChemMatrix as solid support. The peptide chain was assembledusing HBTU as coupling reagent. For Fmoc-deprotection, a 3′ treatmentwith 40% piperidine in DMF was used, followed by a 12′ treatment with20% piperidine in DMF. The furan and benzophenone moiety were introducedusing the commercially available unnatural amino acidsFmoc-ß-(2-furyl)-Ala-OH and Fmoc-L-4-Benzoylphenylalanine-OH. Theseamino acids were coupled manually to the peptide chain using COMU ascoupling reagent.

All peptides were biotinylated after automated synthesis using eitherbiotin or biotin-dPEG(4)-COOH, with COMU as coupling reagent in thepresence of DIPEA. Peptides were cleaved off with TFA/TIS/H2O(95/2.5/2.5) during 2 h and precipitated with cold methyl tert-butylether and analysed by RP-HPLC and ESI-MS or MALDI-MS.

HPLC and Mass Analysis

LC-ESI-MS analysis on peptides was performed on an Agilent 1100 SeriesHPLC instrument equipped with a Phenomenex Kinetex C18 column (150mm×4.6 mm, 5 μm) at 35° C., using a flow rate of 1.5 mL/min. The columnwas eluted with a gradient, starting with 100% H₂O containing 5 mMNH4OAC up to 100% acetonitrile. A G1946C ES-MSD mass detector wasdirectly coupled to the column.

RP-HPLC analysis was also performed on an Agilent 1100 Series HPLCinstrument, equipped with a Phenomenex Luna C18(2) column (250 mm×4.6mm, 5 μM) at 35° C., using a flow rate of 1 mL/min. The column waseluted with a gradient, starting with 100% H₂O containing 0.1% TFA up to100% acetonitrile. The collected fractions were subsequently analysed ona Voyager-DE STR Biospectrometry Workstation (Applied Biosystems), withα-cyano-4-hydroxycinnamic acid as matrix.

Cell Culture

All used cell lines were grown in Dulbecco's Modified Eagle Medium, withhigh glucose content (4.5 mg/mL), no pyruvate and supplemented withGlutamax and 10% (v/v) heat inactivated Fetal bovine serum (FBS) plusantibiotics in a humidified atmosphere with 5% CO₂ at 37° C. TheHEK-myc-KISS1R cell line was kindly provided by Kaiser U. B. of theBrigham and Women's Hospital in Boston (Min et al., 2013, Mol.Endocrinol., 28, 16-27).

Crosslinking Experiments Followed by Western Blot

Crosslinking Furan-Peptides to Cellular Receptor in PBS

Cells were seeded on a 56.7 cm² or a 6-well tissue culture treated plateuntil confluency in normal growth medium. Cell culture medium wasreplaced by cold PBS and the cells were incubated with the peptide 2 or4 (1 μM or as indicated) during 1 h at 4° C. In a competitionexperiment, 10 μM (or as indicated) of 1 was added simultaneously. Afterincubation, 1 equivalent of NBS was added and reaction occurred for 1 h.

Crosslinking Benzophenone-Peptide to Cellular Receptor in PBS

Cells were seeded on a 56.7 cm² or a 6-well tissue culture treatedplate. Cell culture medium was replaced by cold PBS and the cells wereincubated with the peptide 3 or 5 (1 μM or as indicated) during 1 h at4° C. In a competition experiment, 10 μM (or as indicated) of 1 wasadded simultaneously. After incubation, cells were irradiated with UVlight (360 nm) for 30 minutes on ice.

Crosslinking Furan-Peptides to Cellular Receptor in Growth Medium

Cells were seeded on a 56.7 cm² or a 6-well tissue culture treated plateuntil confluency and incubated with the peptides during 30 minutes at37° C. in growth medium with indicated % of serum. A concentration of 1μM of the furan-modified peptide was used. When not relying onendogenous cellular oxidation source, furan oxidation was achieved byadding 1 eq. NBS for 30 minutes at 37° C. In a competition experiment,10 μM of 1 was added simultaneously.

Cell Lysis and Western Blot Analysis

After crosslinking, cells were harvested using trypsinization. The cellpellet was washed with PBS and lysed with lysis buffer (1% CHAPS, 7Murea, 2M thiourea, protease inhibitors, 50 mg/mL DTT). The cell lysatewas sonicated and cell debris was removed by centrifugation at 13,000rpm for 10 minutes at 4° C. Protein concentration was determined usingthe Bradford assay (Biorad). 40 μg total protein was analysed on a 10%SDS-polyacrylamide gel. The separated proteins in the gel weretransferred to a nitrocellulose membrane. The membrane was incubatedovernight at 4° C. with rabbit anti-GPR54 and sequentially incubatedwith anti-rabbit IRDye 800 and streptavidin IRDye 680 for 1 h at roomtemperature. Detection involved scanning of the membrane with an OdysseyInfrared Imaging System (LI-COR Biosciences).

Example 1: Synthesis of Furan-Peptides 2, 4, 6 and 7 and Peptides 1, 3and 5

As model system, the kisspeptin-10 peptide (FIG. 4, compound 1) and itsnative membrane receptor G-protein coupled receptor (GPCR) GPR54 (alsocalled AXOR12 or KISS1R) was used. This model is considered aphysiologically relevant model of a low abundant transientprotein-protein complex, since it is shown that in cells the receptorGPR54 undergoes dynamic post-translational modification and both thereceptor and the cell surface receptor-ligand complex turnover rapidly.

The KISS1 gene encodes a 145 amino acid protein that undergoesproteolysis to metastatin and further truncation to 14, 13 or 10 aminoacid containing carboxy-terminal fragments (the so-called kisspeptins),showing biological activity (Kotani et al., 2001, J. Biol. Chem., 276,34631-6; Ohtaki et al., 2001, Nature, 411, 613-7).

In an initial experiment, the different forms of GPR54 in MDA-MB-231, abreast cancer cell line that endogenously expresses GPR54, wereanalysed. Three GPR54 forms were observed with molecular sizes of 37, 54and 72 kDa (FIG. 5, arrows). The largest form represents the matureglycosylated GPR54, present at the cell surface. In the bindingexperiments below, the formation of a ligand-cell surface receptorcomplex, namely a kisspeptin-GPR54 complex, is visualized by probingbiotin (see below for kisspeptin-10 comprising biotin). FIG. 5 furthershows that untreated MDA-MB-231 already contains two endogenousbiotinylated proteins (indicated by diamonds in all figures). It isassumed that these proteins are endogenously biotinylated carboxylaseswith an apparent molecular weight (MW) of ca. 75 kDa (i.e., present at aslightly higher molecular weight than the covalently bound ligand-cellsurface receptor complex (see Example 2)), and of 130 kDa. Thesebackground signals were used as loading control.

Kisspeptin-10 peptide and analogues were chemically synthesized asdescribed in Materials and methods, Peptide synthesis). The HPLCchromatograms and mass spectra of compound 1 (Chemical Formula:C₆₃H₈₃N₁₇O₁₄, Exact Mass: 1301.63 Da, Molecular Weight: 1302.46 Da), 2(Chemical Formula: C₆₉H₉₄N₁₈O₁₇S, Exact Mass: 1478.68 Da, MolecularWeight: 1479.68 Da), 3 (Chemical Formula: C₇₈H_(1oo)N₁₈O₁₇S, Exact Mass:1592.72 Da, Molecular Weight: 1593.83 Da), 4 (Chemical Formula:C₇₁H₉₅N₁₉O₁₆S, Exact Mass: 1501.69 Da, Molecular Weight: 1502.72 Da), 5(Chemical Formula: C₈₀H₁₀₁N₁₉O₁₆S, Exact Mass: 1615.74 Da, MolecularWeight: 1616.87 Da), 6 (Chemical Formula: C₈₀H₁₁₅N₁₉O₂₂S, Exact Mass:1725.82 Da, Molecular Weight: 1726.97 Da), and 7 (Chemical Formula:C₈₀H₁₁₅N₁₉O₂₂S, Exact Mass: 1725.82 Da, Molecular Weight: 1726.97 Da)were obtained and illustrated the quality of compounds 1 to 7 (resultsnot shown). HPLC and mass analysis was performed as described inMaterials and methods, HPLC and mass analysis.

Compound 1 is the wildtype kisspeptin-10 peptide (FIG. 4). Previousalanine scans on kisspeptin-10 revealed the importance of the C-terminusfor receptor binding. Based on these findings, a furan moiety wasintroduced in the N-terminal part: in peptides 2 and 4 Trp3 and Tyr 1were respectively substituted by 2-furyl-L-alanine (FUA) (FIG. 4). Byanalogy, compounds 3 and 5 carry 4-benzoyl-L-phenylalanine (BPA) atthese positions (FIG. 4). The N-termini of the peptides werebiotinylated for easy detection. Compounds 2 to 5 had biotin directlycoupled to the N-terminus. In compound 6, featuring an identicalsequence as peptide 2, the N-terminus was separated from biotin by 4polyethylene glycol (PEG) units (FIG. 4). Compound 7 is a randomizedsequence version of compound 6, still containing a furan moiety atposition 3 (FIG. 4).

The furan-peptides 2 to 6 were used in comparative methods forcrosslinking a cell surface receptor and a ligand.

Comparative Example: Crosslinking Furan-Peptide Kisspeptin-10 and CellSurface Receptor G-Protein Coupled Receptor GPR54 by Adding NBS as anActivation Signal or by Photoaffinity Crosslinking

Comparative crosslinking experiments were performed with thekisspeptin-10 furan-peptides on MDA-MB-231 cells cultured in a confluentlayer. Cells were cultured as described in Materials and methods, Cellculture. The cell medium was replaced with cold phosphate bufferedsaline (PBS) buffer prior to incubating the MDA-MB-231 cells with thefuran-peptides (see Materials and methods, Crosslinking furan-peptidesto cellular receptor in PBS). These are the same reaction conditionsgenerally used for photo-crosslinking on cells to prevent side-reactionsas well as toxicity by heating upon irradiation.

The MDA-MB-231 cells were incubated with peptides 2 and 4 for 1 h.Subsequently, NBS was added to initiate furan oxidation and inducecrosslink formation via this oxidation. After reaction for 1 h, celllysates were prepared for Western Blot analysis and visualization of thereceptor and the covalently bound peptide-receptor complex (seeMaterials and methods, Cell lysis and Western Blot analysis) (FIG. 6 andFIG. 7). In FIG. 6A, blank shows the endogenous receptor signals(arrowheads). In FIG. 6B and FIG. 7, blank shows the background biotinsignals from endogenously biotinylated carboxylases (75 and 130 kDa,diamonds).

Using furan peptide 2, the formation of a ligand-cell surface complexwas observed (based on the biotin label on peptide 2) at approximately72 kDa (FIG. 6B, lane 4, arrow; FIG. 7, lane 1), which was not the casefor the blank experiment (FIG. 6B, blank; FIG. 7, blank). This cellsurface receptor-ligand complex was visualized using fluorescentstreptavidin binding the biotin on the peptide probe.

In order to verify the specificity of the covalent binding, acompetition experiment was performed in which furan-peptide 2 was addedto the cells simultaneously with a higher concentration of the wild typekisspeptin-10 peptide 1 (FIG. 6B, lane 5). This clearly reduced thecrosslinking signal, confirming the formation of a specific covalentbond between the GPR54 receptor and furan-peptide 2. With furan-peptide4 (FUA at position 1), the formation of a covalent bond could not beobserved, neither in the absence or presence of competing WT-peptide(FIG. 6B, lanes 2 and 3).

A further comparative experiment was performed under comparableconditions for the benzophenone-modified kisspeptin peptides. TheBPA-modified peptides 3 and 5 were incubated for 1 h (at the sameconcentration as peptide 2 and 4) with the cultured cells in cold PBSand the cells were irradiated with UV-light (360 nm) to initiatecrosslinking (see Materials and methods, Crosslinkingbenzophenone-peptide to cellular receptor in PBS). Two independentexperiments were performed. With peptide 3, only a faint and lessdiscrete crosslink signal was visible at 72 kDa (FIG. 7, lane 3). Thephoto-crosslinking reaction was less efficient compared withfuran-peptide 2, and more intense background signals were present (FIG.7, lanes 3 and 4). Lower amounts of crosslinked species were formed uponcompetitive incubation of peptide 3 and an excess of wild type peptide 1(FIG. 7, lane 4, and results not shown).

Example 2: Covalently Binding of Furan-Peptide Kisspeptin-10 and CellSurface Receptor G-Protein Coupled Receptor GPR54 by EndogenousActivation According to an Embodiment of the Present Invention

Furan is in se, an essentially stable, non-reactive moiety requiringoxidation to initiate crosslinking Therefore, previously, NBS was addedto the cells with peptides as an activation signal to convert the furanmoiety into an α-β unsaturated aldehyde for crosslink formation.Surprisingly, in a control experiment without any activation signalperformed on living cells, the present inventors also observed a strongcrosslinking signal. This indicated that the furan moiety was activatedby oxidation in the cell culture, likely by an oxidative agent providedby the cells. The present inventors thus demonstrated for the first timechemical crosslinking of a modified ligand on its endogenous unmodifiedreceptor in cells without any form of exogenous intervention (e.g.,chemical activation signal, (UV-)light). The precise nature of theendogenous (cellular) oxidizing source is unknown.

A method for covalently binding a cell surface receptor and a ligandillustrating the present invention was performed as follows. Briefly,the crosslinking experiments were performed with the kisspeptin-10furan-peptides in situ, on MDA-MB-231 cells cultured in a confluentlayer. The method according to an embodiment of the present inventionwas performed by contacting the MDA-MB-231 cells with furan-peptide 6under physiological conditions (normal growth medium containing 10% FBSand 37° C.) for 30 min (see Materials and methods, Crosslinkingfuran-peptides to cellular receptor in growth medium). After reaction,cell lysates were prepared for Western Blot analysis and visualizationof the receptor and the covalently bound ligand-cell surface receptorcomplex (see Materials and methods, Cell lysis and Western Blotanalysis). Using furan-peptide 6, the formation of a crosslink atapproximately 72 kDa was observed (FIG. 8, lane 2, arrow), which was notthe case for the blank experiment (FIG. 8, blank). The ligand-cellsurface receptor complex was visualized using fluorescent streptavidinbinding the biotin on the peptide probe. In FIG. 8, only the signal forbiotin is shown. The efficiency of covalently binding of furan-peptide 6was shown to be similar to that of furan-peptide 2 (FIG. 9B).Furan-peptide 6 contains a short polyethylene glycol linker of fourunits between the N-terminus and the biotin moiety. Efficiency ofcovalently binding of furan-peptide 6 was compared to that offuran-peptide 2 in medium comprising serum at 37° C. A slightly strongersignal of the covalent cell surface receptor-ligand complex was observedwhen using furan-peptide 6 (FIG. 9B). Better accessibility of thestreptavidin to the biotin moiety due to less steric hindrance is apossible explanation for this finding. The 72 and 54 kDa subspecies ofthe GPCR54 receptor are shown in a FIG. 9A (arrowheads).

The covalently binding was also performed under the same conditions butwith the addition of NBS as an activation signal (FIG. 8, lane 1).Higher efficiency was seen without addition of any exogenous activationsignal to the medium (FIG. 8, compare lane 1 and lane 2). Hence, themethod according to an embodiment of the present invention allows forefficient covalently binding of a cell surface receptor and a ligandwithout the need for any chemical or physical activation signal.

The molecular weight of the biotinylated covalently bound cell surfacereceptor-ligand complex indicates that the furan-peptide 6 mainlycrosslinks to the membrane-presented, glycosylated receptor form (FIG.5). This illustrates that the covalent binding of the kisspeptin-10 andGPR54 occurs in situ at the plasma membrane and thus at the site of thephysiological ligand interaction.

To give final proof to the formation of a selective covalent bond usingthe method according to an embodiment of the present invention, we usedfuran-peptide 7, a random kisspeptin-10, with a furan moietyincorporated at the third position. Like furan-peptide 6, this peptidewas biotinylated using a PEG(4)-linker at the N-terminus. Uponincubation, absolutely no crosslink signal was observed (FIG. 10B, lane3). The 72 kDa form of the GPCR54 receptor is shown in a FIG. 10A(arrowheads).

The results thus illustrate that the method according to an embodimentof the present invention allows covalent binding upon specific bindingof a ligand to its cell surface receptor.

Example 3: Covalent Binding of a Cell Surface Receptor and a LigandSpecifically Binding to the Cell Surface Receptor in Different CellLines According to Embodiments of the Present Invention

To check the applicability of the method according to the presentinvention in different biological contexts, different cell linesexpressing GPR54 receptor were used. In human cancer cell lines HeLa(FIG. 12A and FIG. 12B) and MCF-7 (FIG. 13A and FIG. 13B), efficientcrosslinking of furan-peptide 6 and GPR54 was achieved upon incubatingthe living cells with furan-peptide 6 (FIG. 12B and FIG. 13B,respectively, arrow). MDA-MB-231, HeLa and MCF-7 are all human breastcancer cell lines. Furthermore, it was tested whether the covalentbinding was also observed using normal cell lines, for example cellslines derived from non-diseased such as non-cancerous or non-infectedtissue or cells.

In mouse fibroblast NIH 3T3 cell line, covalent binding of furan-peptide6 and GPR54 was also efficiently achieved upon incubation of the livingcells with furan-peptide 6 (FIG. 11B, crosslink indicated by arrow). Inall tested cell lines, no exogenous intervention such as NBS treatmentwas needed for the formation of the covalent bonds. Interestingly, inNIH 3T3, the inherent relative abundance of the different receptorspecies is very different: the GPR54 signal at 54 kDa was barely visible(FIG. 11A) in contrast to a very strong band at 72 kDa (FIG. 11B). Asthe furan modified kisspeptin-10 peptide preferably crosslinks with this72 kDa receptor form, a very strong crosslinking signal was observed(FIG. 11B, lane 2).

Finally, the human embryonic kidney cell line, HEK-myc-KISS1R, thatstably expresses relatively low levels of myc-tagged GPR54 was used.Furan-peptide 6 was tested with the addition of NBS and without theaddition of any activation signal (FIG. 14A and FIG. 14B). In bothconditions, covalent binding to the cell surface receptor GPR54 wasobserved (FIG. 14B), but the crosslink was stronger when no activationsignal was added (FIG. 14B, compare lanes 2 and 3).

Example 4: Covalent Binding of a Cell Surface Protein and a LigandSpecifically Binding to the Cell Surface Protein In Vivo According to anEmbodiment of the Present Invention

To test the applicability of the method according to the presentinvention in vivo, MDA-MB-231 cells are injected subcutaneously intomice (three groups of n=3). When tumours reach a size of around 250-500mm³ (estimated after approximately 10-15 days), injections in the tumourare performed (intra-tumour injection) with the furan-kisspeptin peptide6 (FIG. 4) (group 1, n=3), with peptide 7 (FIG. 4) (group 2, n=3), or amock injection is performed (group 3, i.e. control group, n=3). Peptide7 is a randomized sequence version of peptide 6 still containing a furanmoiety at position 3. The peptide concentration in the injectedphysiological buffer is minimally 1 μM. To estimate the maximal peptideconcentration, toxicity tests are performed on cultured cells in vitrousing peptide 6 and 7, and acute local toxicity tests are performedusing subcutaneous injection of peptide 7 in mice. The injected peptides6 and 7 also comprise biotin (FIG. 4) to allow detection for instance byWestern blot analysis. The peptides may further comprise a fluorescentagent for immunohistochemistry. Several hours after injection, thetumour is isolated, proteins are extracted from the tumour tissue, andcovalent binding of the kisspeptin peptide to its cell surface receptorGPR54 is analysed by Western blot analysis. Covalent binding of the cellsurface protein (GPR54) to the ligand (kisspeptin peptide) is detected.Alternatively, the tumour tissue is prepared for immunohistochemistry.

Example 5: Covalent Binding of a Cell Surface Protein and a LigandSpecifically Binding to the Cell Surface Protein According to anEmbodiment of the Present Invention to Allow In Situ Visualization ofthe Cell Surface Protein on Adherent Cells

In order to study the dynamics of a cell surface receptor such as C—X—Cchemokine receptor type 4 (CXCR4) in the cell membrane, a set of CVX15peptides is synthesized, each carrying one furan moiety at a differentposition within the sequence. CVX15 is a peptide antagonist of theG-protein coupled receptor CXCR4. CVX15 is a 16-residue cyclic peptidecharacterized as an HIV-inhibiting and anti-metastatic agent and itsbinding site on the CXCR4 receptor has been determined (Wu et al, 2010,Science, 330, 1066-71). When synthesizing the set of CVX15 peptides,each carrying one furan moiety at a different position within thesequence; the choice of positions can be guided by the information ofthe known binding site. The furan-containing CVX15 peptides are inaddition tagged with a fluorescent moiety which is not interfering withbinding.

Adherent living MDA-MB-231 cells (e.g., 5×10⁵ cells) are incubated incell culture medium with each of the different furan-containing CVX15peptides (concentration between 0.1-5 μM) to allow crosslinking Usingconfocal microscopy on fixed cells, the furan-containing CVX15 peptidethat most efficiently crosslinks to CXCR4, is selected for furtherexperiments. Subsequently, living MDA-MB231 cells are contacted with theselected furan-containing CVX15 peptide (i.e., a first furan-containingCVX15 peptide containing a fluorescent moiety) to covalently bind thepeptide to CXCR4 on the cell surface. After washing away unboundfuran-containing CVX15 peptide, the dynamics of the CXCR4 receptor onthe cell surface is visualized and recorded using live cell imaging viathe fluorescent moiety coupled to the furan-containing CVX15 peptide. Inaddition, the selected furan-containing CVX15 peptide is synthesized butcarrying a different fluorophore (i.e., a second furan-containing CVX15peptide). MDA-MB cells are contacted with a mixture of the first andsecond furan-containing CVX15 peptides, each carrying a differentfluorophore, thereby obtaining CXCR4 receptors covalently bound to CVX15peptides carrying different fluorophores. CXCR4 receptor clustering onthe cell surface of living MDA-MB-231 cells is detected based onco-localization of the two fluorophore signals.

Example 6: Covalent Binding of a Cell Surface Protein and a LigandSpecifically Binding to the Cell Surface Protein According to anEmbodiment of the Present Invention to Allow In Situ Visualization ofthe Cell Surface Protein on Cells in Suspension

A set of furan-containing CVX15 peptides is prepared as described inExample 5. A Jurkat cell line (i.e., T-cell cell line growing insuspension culture) is used. Living Jurkat cells (1×10⁵ cells) arecontacted with each of the different furan-containing CVX15 peptides(concentration between 0.1-5 μM) to allow covalent binding. In order toallow imaging, the cells are embedded in a small volume (e.g. 20-40 μl)of a hydrogel composed of extracellular matrix proteins (e.g., collagentype I). The furan-containing CVX15 peptide that most efficientlycrosslinks to CXCR4 is selected using confocal microscopy on fixedcells. Next, living Jurkat cells are contacted with the selectedfuran-containing CVX15 peptide to form the fluorescent ligand-receptorcovalently bound complex. The cells are then embedded in a small volume(20-40 μl) of a hydrogel composed of extracellular matrix proteins (e.g.collagen type I) to allow imaging. The dynamics of the CXCR4 receptor,covalently bound to the fluorescent CVX15 ligand, on the cell surface isvisualized and recorded using live cell imaging. In addition, theselected furan-containing CVX15 peptide is synthesized but carrying adifferent fluorophore (i.e., a second furan-containing CVX15 peptide).Jurkat cells are contacted with a mixture of the first and secondfuran-containing CVX15 peptides, each carrying a different fluorophore,thereby obtaining CXCR4 receptors covalently bound to CVX15 peptidescarrying different fluorophores. CXCR4 receptor clustering on the cellsurface of living Jurkat cells is detected based on co-localization ofthe two fluorophore signals.

Example 7: Covalent Binding of a Cell Surface Protein and a LigandSpecifically Binding to the Cell Surface Protein According to anEmbodiment of the Present Invention to Allow In Situ Visualization ofthe Cell Surface Protein

In order to study the dynamics of a cell surface receptor such as C—Cchemokine receptor type 5 (CCR5) in the cell membrane, a set ofMaraviroc compounds is synthesized, each carrying one furan moiety at adifferent position. Maraviroc is a small molecule antagonist of the CCR5cell surface receptor. The Maraviroc binding site on the CCR5 receptorhas been determined (Tan et al., 2013, Science, 341, 1387-90). Bychemical synthesis, a set of Maraviroc compounds is generated, eachcompound comprising a fluorescent moiety and carrying one furan moietyat a different position. The choice of positions is guided by theinformation of the known binding site. Living cells (1×10⁵ cells) of theJurkat cell line in suspension culture are incubated, each with aMaraviroc compound (concentration e.g. between 0.1-10 μM) to allowcrosslinking. To allow imaging, the cells are embedded in a small volume(e.g. 20-40 μl) of hydrogel composed of extracellular matrix proteins(e.g. collagen type I). The furan-containing Maraviroc compound thatmost efficiently crosslinks to the cell surface receptor, is selectedusing confocal microscopy on fixed cells. Subsequently, living Jurkatcells are contacted with the selected furan-containing Maraviroccompound to form the fluorescent ligand-receptor covalently boundcomplex. The cells are embedded in a small volume (20-40 μl) of hydrogelcomposed of extracellular matrix proteins (e.g. collagen type I) inorder to allow imaging. The dynamics of the CCR5 receptor, covalentlybound to the fluorescent Maraviroc ligand, on the cell surface isvisualized and detected using live cell imaging.

Example 8: Cell-Based Assay for Identifying for an Orphan Ligand a CellSurface Protein being Capable of Specifically Binding to the LigandAccording to an Embodiment of the Present Invention

A cell-based assay is performed to identify the cell surface protein foran orphan ligand, such as the alpha fetoprotein peptide (AFPep) ligand(also called growth inhibitory protein 8 or GIP-8). This peptide ligandis a 9-mer circularized variant of an 8-mer peptide derived from alphafetoprotein (AFP). AFPep activities in vitro and in vivo includespecific effects on estrogen receptor (ER) positive cancer cells. Thereceptor of AFPep ligand is unknown. Variants of the 9-mer AFPep, and ofits non-circular 8-mer peptide, carrying one furan moiety (either at theN- or C-terminus or at an internal position) and a label for detection(e.g., biotin) are synthesized. Living ER-positive breast cancer cellline MCF-7 cells (5×10⁶ cells) are contacted with each of these peptides(e.g. at 0.5 to 2 μM) in cell medium. The ER-negative cell lineMDA-MB-231, for which it is known that AFPep causes no growth inhibitoryeffects, and thus anticipated not to express the unknown cell surfacereceptor, is used as negative control. The cells are harvested, lysed,and analysed using Western Blotting with an antibody against the labelto detect the presence of a covalently bound complex in which thepeptide ligand is specifically bound to a cell surface protein. Massspectrometry analysis is used to identify the cell surface receptorcovalently bound to AFPep.

Example 9: Cell-Based Assay for Identifying a Binding Site of a CellSurface Protein and a Ligand Specifically Binding the Cell SurfaceProtein According to an Embodiment of the Present Invention

A cell-based assay is performed to identify a binding site of a cellsurface protein and a ligand such as the alpha fetoprotein peptide(AFPep) ligand, as described in Example 8. Variants of the non-circular8-mer peptide variant of AFPep carrying one furan moiety and a biotinlabel are used to identify the binding site. Living ER-positive breastcancer cell line MCF-7 cells (5×10⁶ cells) are contacted with thesepeptides (e.g. at 0.5 to 2 μM) in cell medium. The ER-negative cell lineMDA-MB-231, for which it is known that the AFPep causes no growthinhibitory effects, and is thus anticipated not to express the receptor,is used as negative control. The cells are harvested, lysed, andanalysed using Western Blotting using an antibody against the label todetermine the presence of a covalently bound complex in which thepeptide ligand is specifically bound to a cell surface protein. Thepresence of a covalently bound complex identifies the amino acidcomprising a furan moiety as being part of the binding site of the cellsurface protein and the peptide. In addition or alternatively, massspectrometry analysis is used to identify the covalently bound cellsurface receptor of AFPep and eventually to confirm the binding site.

Example 10: Cell-Based Assay for Identifying for an Opioid Peptide, theOpioid Receptor (Subtype) Capable of Specifically Binding to the OpioidPeptide According to an Embodiment of the Present Invention

A first goal of this experiment was the synthesis of a furan-modifiedopioid peptide (e.g. receptor agonist peptide). As the N-terminus of theopioid peptide (H—Y-r-F—F—NH₂ with r=d-Arginine) (FIG. 15, wild typepeptide or “WTP”) is known to be crucial for binding with the opioidreceptor, the C-terminus of the peptide (i.e. C-terminal amide) waschosen for modification. A furan moiety directly at the C-terminus maygive rise to a more troublesome synthesis, and for that reason an extraglycine was attached to the C-terminus as linker (FIG. 15, “WTP-G”).Different opioid peptide analogues were produced to be tested for theirability to bind the opioid receptor (FIG. 15, “FUA3”, “FUA-G”, and“FUA4”).

Next, the synthesised peptides were subjected to biological activitytesting, to know whether they still bind to the opioid receptor. If so,it is tested to which opioid receptor subtype they bind. It is alsoinvestigated whether the opioid peptides behave as agonist orantagonist. The following assays are performed: radio-ligand bindingassay, ³⁵S GTP binding assay, and tissue experiments, including guineapig ileum (GPI) and mouse vas deferens (MVD) tests.

TABLE 1 Opioid receptor binding affinities of irreversible orthostericligands to the human opioid receptors (CHO-hOR cells) Binding affinityK_(i) (nM) ± SEM Peptide μ-opioid receptor (MOR) WTP-G 5.84 ± 1.29 WTP8.04 ± 0.44 FUA3   32.6 FUA-G 145 FUA4 251

Based on these results, the binding of the furan-modified peptide FUA3was acceptable. Further biological assays are performed.

The furan-modified opioid peptides that bind to opioid receptors arechosen for further crosslinking experiments. Cells overexpressing anopioid receptor subtype are incubated with the furan-modified opioidpeptides without the addition of an exogenous activation signal,illustrating an embodiment of the invention. Crosslinking with theopioid receptor subtypes is evaluated by Western blotting. Ifcrosslinking is successful, the crosslinked complex can be used toelucidate the opioid peptide-opioid receptor complex.

The invention claimed is:
 1. A method for covalently binding a cellsurface protein and a ligand, the ligand being capable of specificallybinding to the cell surface protein, the method comprising contactingliving cells expressing the cell surface protein with the ligandcomprising at least one furan moiety without the addition of anexogenous activation signal, thereby covalently binding the cell surfaceprotein and the ligand.
 2. The method according to claim 1, wherein thecell surface protein is selected from the group consisting of a cellsurface receptor, a cell adhesion molecule, and a cell surface protease.3. The method according to claim 1, wherein the cell surface protein isa cell surface receptor selected from the group consisting of aG-protein coupled receptor (GPCR), an immune receptor, an ionchannel-linked receptor, and an enzyme-linked receptor preferablywherein the cell surface protein is a GPCR.
 4. The method according toclaim 1, wherein the ligand is a peptide, a nucleoside, a nucleic acid,a lipid, a polysaccharide, a small molecule, or a combination thereof,preferably wherein the ligand is a peptide.
 5. The method according toclaim 1, wherein the cell surface protein is a GPCR and the ligand is apeptide.
 6. The method according to claim 1, wherein the furan moiety ofthe ligand is oxidized by endogenous activation.
 7. The method accordingto claim 6, wherein the endogenous activation occurs at theextracellular space of the cell membrane.
 8. The method according toclaim 1, wherein the cell surface protein comprises at least one aminegroup, hydroxyl group, sulfhydryl group, imidazole group and/or indolegroup.
 9. The method according to claim 6, wherein the oxidized furanmoiety of the ligand reacts with the amine group, hydroxyl group,sulfhydryl group, imidazole group and/or indole group of the cellsurface protein.
 10. The method according to claim 1, wherein the methodis performed under physiological conditions.
 11. The method according toclaim 1, wherein the living cells are normal cells.